Enzymatic Production Of Peracids Using Perhydrolytic Enzymes

ABSTRACT

A process is provided to produce a concentrated aqueous peracid solution in situ using at least one enzyme having perhydrolase activity in the presence of hydrogen peroxide (at a concentration of at least 500 mM) under neutral to acidic reaction conditions from suitable carboxylic acid esters (including glycerides) and/or amides substrates. The concentrated peracid solution produced is sufficient for use in a variety of disinfection and/or bleaching applications.

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/413,246 filed Apr. 28, 2006, which claims the benefit ofU.S. Provisional Application No. 60/676,116 filed Apr. 29, 2005.

FIELD OF THE INVENTION

This invention relates to the field of organic peracid synthesis andenzyme catalysis.

BACKGROUND OF THE INVENTION

Peracids have been used for disinfection in a number of applications.U.S. Pat. No. 6,545,047 B1 describes a method for sanitizing animalcarcasses using antimicrobial compositions containing one or moreperacids. U.S. Pat. No. 6,183,807 B1 describes a method for cleaning andsanitizing meat products using antimicrobial compositions containing oneor more peracids. U.S. Pat. No. 6,518,307 B2 describes a method forcontrolling microbial populations in the gastrointestinal tract ofanimals by orally administering an effective amount of peracid. U.S.Patent Application Publication No. 20030026846 A1 describes a method ofusing peracid/acid compositions to control pathogenic organisms onliving plant tissue. U.S. Pat. No. 5,683,724 describes a process forpreventing microbial growth in aqueous streams used for transporting orprocessing food products and packaged foods that uses an effectiveantimicrobial concentration of peracid.

Peracids can be prepared by the chemical reaction of a carboxylic acidand hydrogen peroxide (see Organic Peroxides, Daniel Swern, ed., Vol. 1,pp 313-516; Wiley Interscience, New York). The reaction is usuallycatalyzed by a strong inorganic acid, such as concentrated sulfuricacid. The reaction of hydrogen peroxide with a carboxylic acid is anequilibrium reaction, and the production of peracid is favored by theuse of an excess concentration of peroxide and/or carboxylic acid, or bythe removal of water. There are several disadvantages to the chemicalreaction for peracid production: a) the high concentration of carboxylicacid used to favor production of peracid can result in an undesirableodor when using the peracid-containing solution, 2) the peracid isoftentimes unstable in solution over time, and the concentration ofperacid in the solution decreases during storage prior to use, and 3)the formulation is often strongly acidic due to the use of aconcentrated sulfuric acid as catalyst. One way to overcome thedisadvantages of the chemical production of peracids is to employ anenzyme catalyst in place of a strong acid catalyst. The use of an enzymecatalyst allows for the rapid production of peracid at the time of use,avoiding the problem of storage of peracid solutions and of usingchemically-produced peracid solutions containing an unknownconcentration of peracid. The high concentrations of carboxylic acidstypically used to produce peracid via the direct chemical reaction withhydrogen peroxide are not required for enzymatic production of peracid,where the enzyme-catalyzed reaction can use a carboxylic acid ester oramide as substrate at a much lower concentration than is typically usedin the chemical reaction. The enzyme reaction can be performed across abroad range of pH, dependent on enzyme activity and stability at a givenpH, and on the substrate specificity of the enzyme for perhydrolysis ata given pH.

Enzymes can catalyze the perhydrolysis of esters and amides to producethe corresponding peroxycarboxylic acids (Equations 1 and 2), however,most known methods for preparing peracids from the correspondingcarboxylic acid esters or amides using enzyme catalysts do not produceand accumulate a peracid at a sufficiently-high concentration to beefficacious for disinfection in a variety of applications.

The use of hydrogen peroxide as an enzyme substrate for the enzymaticperhydrolysis of carboxylic acid esters or amides can be problematic, ashydrogen peroxide is known to oxidatively inactivate numerous enzymes(M. R. Gray, Biotech Adv., 7:527 (1989)). K. Kleppe (Biochemistry, 5:139(1966)) report that hydrogen peroxide inactivates enzymes by modifyingcertain amino acid residues in proteins, where at acid pH valuesmethionine is easily oxidized to methionine sulfoxide, and at basic pHvalues tryptophan is destroyed. D. A. Estell et al. (J. Biol. Chem.,260:6518 (1985)) describe inactivation of enzymes containing methionine,cysteine or tryptophan residues by hydrogen peroxide, anddemonstrate >80% inactivation of the protease subtilisin from Bacillusamyloliquefaciens in less than 6 minutes or 4 minutes using 0.1 M or 1.0M hydrogen peroxide, respectively. Inactivation of peroxidases by 5 mMto 50 mM hydrogen peroxide is reported by M. B. Arnao et al. (Biochim.Biophys. Acta, 1041:43 (1990)), and B. Valderrama et al. (Chemistry &Biology, 9:555 (2002)) review the inactivation of peroxidases byoxidative species such as hydrogen peroxide. P. F. Greenfield et al.(Anal. Biochem., 65:109 (1975)) report an increase in inactivation ofglucose oxidase with increasing hydrogen peroxide concentration. For theconversion of cephalosporin C to 7-aminocephalosporanic acid, both aD-amino acid oxidase and a glutaryl acylase were inactivated by thebyproduct hydrogen peroxide produced by the oxidase (F. Lopez-Gallego,et al., Adv. Synth. Catal., 347:1804 (2005)). In view of these and otherteachings, previously reported methods for enzymatic production ofperacid utilize low concentrations of added hydrogen peroxide, where alow concentration of hydrogen peroxide would be expected to reduce orlimit enzyme inactivation during the perhydrolysis reaction.

U.S. Pat. No. 3,974,082 (“the '082 patent”) describes the production ofbleaching compositions for laundry detergent applications by contactingthe material to be bleached with an aqueous solution containing anoxygen-releasing inorganic peroxygen compound, an acyl alkyl ester, andan esterase or lipase capable of hydrolyzing the ester. The bleachingcompositions cited in the '082 patent are highly alkaline (using suchbuffering agents as pentasodium tripolyphosphate or sodium carbonate),and no data is presented for either the concentration of peracidsproduced in the cited compositions, or for the utility of the citedcompositions for bleaching of laundry. The bleaching compositions citedin the '082 patent contain up to 40% by weight of per-compound, forexample, hydrogen peroxide or alkali metal salts of percarbonate,perborate, persilicate and perphosphate. The bleaching compositions areadded to water in amounts up to 12.5 grams per liter of water toinitiate the enzyme-catalyzed perhydrolysis reaction, where the maximumconcentration of hydrogen peroxide present in the enzyme-catalyzedperhydrolysis reaction is 5 grams/liter, equivalent to ca. 147 mMhydrogen peroxide.

U.S. Pat. No. 5,296,161 (“the '161 patent”) describes an activatedoxidant system providing enhanced stain removing ability in both highand low temperature wash applications. The oxidant system is capable ofin situ generation of >0.1 ppm peracid by enzymatic perhydrolysis, wherein the absence of added enzyme the ester substrate is incapable ofsubstantial chemical perhydrolysis. The oxidant system uses a source ofperoxygen, a lipase or esterase, and glycerides or monoacylated ethyleneglycol or propylene glycol derivatives to generate peracid. Themost-preferred enzyme substrate in the '161 patent oxidant system iseither trioctanoin or tridecanoin, the enzymatic reaction is carried outat a pH of from 7.5 to 11.0, and none of the accompanying examplesdemonstrate the production of greater than 10 ppm of peracid. Thehighest concentration of hydrogen peroxide present in the exemplifiedperhydrolysis reactions was 1314 ppm, equivalent to ca. 38.6 mM H₂O₂.

U.S. Pat. No. 5,364,554 describes an activated oxidant system for insitu generation of peracid in aqueous solution using a protease enzyme,a source of hydrogen peroxide, and an ester substrate that is preferablychemically non-perhydrolyzable. A method of bleaching and a method offorming peracid are also disclosed. The enzymatic reactions are carriedout at a pH of from about 8.0 to 10.5, and none of the accompanyingexamples demonstrate the production of greater than 5 ppm of peracid.The concentration of hydrogen peroxide present in the exemplifiedperhydrolysis reactions was 400 ppm, equivalent to ca. 11.8 mM H₂O₂.

O. Kirk et al. (Biocatalysis, 11:65-77 (1994)) investigated the abilityof hydrolases (lipases, esterases, and proteases) to catalyzeperhydrolysis of acyl substrates with hydrogen peroxide to formperoxycarboxylic acids, and reported that perhydrolysis proceeds with avery low efficiency in aqueous systems. Furthermore, they found thatlipases and esterases degraded percarboxylic acid to the correspondingcarboxylic acid and hydrogen peroxide. They also found that proteasesneither degraded nor catalyzed perhydrolysis of carboxylic acid estersin water. The authors concluded that esterases, lipases and proteasesare, in general, not suitable for catalyzing perhydrolysis of simpleesters, such as methyl octanoate and trioctanoin, in an aqueousenvironment.

The problem to be solved is to provide an aqueous enzymatic process forin situ production of peracid compositions under neutral to acidicconditions from non-toxic and inexpensive carboxylic acid esters,amides, and/or glycerides at concentrations suitable for use as adisinfectant or bleach in a variety of applications. Preferably, theprocess will produce a concentrated aqueous solution of peracid withinat least about 5 minutes. As such, the enzymatic perhydrolysis processshould occur in the presence of at least 500 mM hydrogen peroxide(peroxygen source). It has been reported that for certain disinfecting,cleaning or bleaching applications a non-alkaline peracid solution ispreferred. As such, the process should produce an aqueous peracidsolution in a single step under neutral to acidic conditions, morepreferably under acidic conditions. The process preferably needs toproduce peracid compositions comprised of at least 10 ppm peracid (forexample peracetic acid), more preferably at least 100 ppm, and even morepreferably in the range of about 100 to about 5000 ppm, where theresulting peracid composition can be used directly, or diluted to thedesired concentration of peracid prior to use, to produce a 5-log or6-log reduction in the concentration of the targeted infectiousmicroorganism in about 5 minutes to about 10 minutes, at temperaturesranging from about 0° C. to about 60° C., preferably about 4° C. toabout 30° C., most preferably about 10° C. to about 25° C.

A second problem to solve is to provide a process to produce amulti-functional composition that has disinfecting, bleaching andprion-degrading activity. The process should produce an aqueous peracidsolution in situ comprising a sufficient disinfecting and/or bleachingconcentrations of peracid and one or more prion-degrading proteases.

An additional problem to be solved is the lack of a combination ofenzyme catalyst and enzyme substrate that results in the conversion ofcarboxylic acid esters or amides to percarboxylic acid at aconcentration more efficacious for bleaching in laundry applications,compared to the concentrations of peracids previously disclosed in theprior art. A solution to the problem needs to 1) efficiently produce anaqueous solution of peracid where the peracid is present in sufficientconcentration to act as a disinfectant or bleaching agent, 2) use anenzyme catalyst having suitable perhydrolase activity for convertingcarboxylic acid esters, amides, and/or glycerides to the correspondingperacids in aqueous solution; 3) provide methods to improve catalyststability to increase the catalyst productivity, thereby decreasingcatalyst cost; and 4) provide methods to efficiently and economicallyobtain peracids from relatively inexpensive and non-toxic startingmaterials.

SUMMARY OF THE INVENTION

The stated problems have been solved by providing a process to producean aqueous peracid solution in situ using at least one enzyme havingperhydrolase activity in the presence of hydrogen peroxide (at aconcentration of at least 500 mM) under neutral to acidic reactionconditions from suitable carboxylic acid esters (including glycerides)and/or amides substrates. The concentration of peracid produced issufficient for use in a variety of disinfection and/or bleachingapplications.

One aspect of the invention provides a process for producing aconcentrated aqueous peracid solution comprising;

-   -   a) providing a set of peracid reaction components, said        components comprising:        -   1) at least one substrate selected from the group consisting            of:            -   i) esters having the structure

-   -   -   -   wherein R₁=C1 to C10 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₂=C1 to C10 straight chain or                branched chain alkyl group, (CH₂CH₂—O)_(n)H or                (CH₂CH(CH₃)—O)_(n)H and n=1 to 10;            -   ii) glycerides having the structure

-   -   -   -   wherein R₁=C1 to C10 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₃ and R₄ are individually H or                R₁C(O); and            -   iii) amides having the structure:

-   -   -   wherein R₅ and R₆=H or a C1 to C5 straight chain or branched            alkyl group;        -   2) a source of peroxygen that provides a concentration of at            least 500 mM hydrogen peroxide upon combining said reaction            components; and        -   3) at least one enzyme catalyst having perhydrolase            activity, wherein said enzyme catalyst is selected from the            group consisting of lipases, esterases, proteases, and            mixtures thereof; and

    -   b) combining said reaction components at a pH of about 2.5 to        about 7.5, whereby a concentrated peracid solution is produced        within at least about 5 minutes to about 2 hours after combining        said reaction components.

This process produces a concentrated peracid solution with a peracidconcentration of at least 10 ppm.

In one aspect of the invention the at least one substrate is an estersubstrate selected from the group consisting of methyl lactate, ethyllactate, methyl glycolate, ethyl glycolate, methyl methoxyacetate, ethylmethoxyacetate, methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, andmixtures thereof.

In another aspect of the invention the at least one substrate is aglyceride substrate selected from the group consisting of monoacetin,diacetin, triacetin, monobutyrin, dibutyrin, tributyrin, glycerylmonooctanoate, glyceryl dioctanoate, glyceryl trioctanoate, and mixturesthereof.

In yet another aspect of the invention the at least one substrate is aglyceride substrate is selected from the group consisting of monoacetin,diacetin, triacetin, and mixtures thereof.

In one aspect of the invention the at least one enzyme catalyst is atleast one lipase produced by an organism selected from the generaAspergillus, Rhizopus, Penicillium, Candida, Pseudomonas, Mucor,Thermomyces, Alcaligenes, and Sus.

In another aspect the enzyme catalyst is at least one lipase selectedfrom the group consisting of Candida antartica lipase B and Aspergillusniger lipase.

In another aspect of the invention the at least one enzyme catalyst isat least one protease with perhydrolytic activity and prion-degradingactivity.

In another aspect of the invention the enzyme catalyst includes at leastone lipase and at least one prion-degrading protease.

In many aspects of the invention the peracid produced by combination ofthe reaction components is peracetic acid.

Another aspect of the invention provides a method for disinfecting alocus having a concentration of microorganisms or viruses orcombinations thereof, said method comprising:

-   -   a) providing a set of peracid reaction components, said        components comprising:        -   1. at least one substrate selected from the group consisting            of:            -   i) esters having the structure

-   -   -   -   wherein R₁=C1 to C10 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₂=C1 to C10 straight chain or                branched chain alkyl group, (CH₂CH₂—O)_(n)H or                (CH₂CH(CH₃)—O)_(n)H and n=1 to 10;            -   ii) glycerides having the structure

-   -   -   -   wherein R₁=C1 to C10 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₃ and R₄ are individually H or                R₁C(O); and            -   iii) amides having the structure:

-   -   -   wherein R₅ and R₆=H or a C1 to C5 straight chain or branched            alkyl group;        -   2) a source of peroxygen that provides a concentration of at            least 500 mM hydrogen peroxide upon combining said reaction            components; and        -   3) at least one enzyme catalyst having perhydrolase            activity, wherein said enzyme catalyst is selected from the            group consisting of lipases, esterases, proteases, and            mixtures thereof;

    -   b) combining said reaction components at a pH of 2.5 to 7.5,        whereby a concentrated aqueous peracid solution is formed having        a peracid concentration of at least 10 ppm within at least about        5 minutes to about 2 hours of combining said reaction        components;

    -   c) optionally diluting the said aqueous peracid solution; and

    -   d) contacting said locus with the aqueous peracid solution        produced in step b) or step c) whereby the concentration of said        microorganisms is reduced at least 3-log.

In another aspect of the invention the locus is contacted with theaqueous peracid solution produced in step b) or step c) as describedabove, within about 48 hours of combining said reaction components, orwithin about 5 minutes to about 48 hours.

Another aspect of the invention provides a method for decontaminating ordisinfecting a locus contaminated with one or more pathogens includingan infective prion or prion particle comprising

-   -   a) providing a set of peracid reaction components, said        components comprising:        -   1) at least one substrate selected from the group consisting            of:            -   i) esters having the structure

-   -   -   -   wherein R₁=C1 to C10 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₂=C1 to C10 straight chain or                branched chain alkyl group, (CH₂CH₂—O)_(n)H or                (CH₂CH(CH₃)—O)_(n)H and n=1 to 10;            -   ii) glycerides having the structure

-   -   -   -   wherein R₁=C1 to C10 straight chain or branched chain                alkyl optionally substituted with an hydroxyl or a C1 to                C4 alkoxy group and R₃ and R₄ are individually H or                R₁C(O); and            -   iii) amides having the structure:

-   -   -   wherein R₅ and R₆=H or a C1 to C5 straight chain or branched            alkyl group;        -   2) a source of peroxygen that provides a concentration of at            least 500 mM hydrogen peroxide upon combining said reaction            components; and        -   3) at least one enzyme catalyst having perhydrolase            activity, wherein said enzyme catalyst is selected from the            group consisting of lipases, proteases, and mixtures            thereof;        -   4) at least one prion-degrading protease wherein one or more            of the prion-degrading proteases may be the same as the            enzyme catalyst providing the perhydrolase activity in            (a)(3);

    -   b) combining said reaction components at a pH of 2.5 to 7.5,        whereby a concentrated peracid solution is produced having a        peracid concentration of at least 10 ppm within at least about 5        minutes to about 2 hours of combining said reaction components;        and

    -   c) optionally diluting said peracid solution produced in step        (b).

    -   d) contacting a locus contaminated with a microorganism, a        virus, a prion or prion particle, or a combination thereof with        the aqueous peracid solution produced in step b) or step c)        whereby said locus is disinfected and said prion particle is        degraded.

Another aspect of the invention provides a concentrated peracid solutionproduced in situ by the process described above, wherein said solutionincludes at least one protease suitable for prion degradation.

Yet another aspect of the invention provides a multifunctionaldisinfectant composition comprising the concentrated peracid solutionproduced by the process described above, wherein said composition issuitable for prion degradation, use as a biocide, use as a virucide, orcombinations thereof.

In yet another embodiment, a biocidal composition having prion-degradingactivity and components suitable for producing an effective amount ofperacid in situ is also provided, said composition comprising componentssuitable for producing peracid in situ, wherein said components compriseone or more prion-degrading enzymes, a suitable substrate, at least oneenzyme having perhydrolase activity, and a source of peroxygen.

DETAILED DESCRIPTION OF THE INVENTION

The stated problems have been solved by the discovery of a combinationof enzymes and carboxylic acid esters or amides that, in the presence ofan inorganic source of peroxygen (for example, hydrogen peroxide),produce concentrations of peracids sufficient for disinfection orbleaching applications. It was unexpected that concentrations ofhydrogen peroxide of from about 500 mM to about 2500 mM could beemployed in enzyme-catalyzed perhydrolysis reactions to generate peracidin concentrations of as high as 5000 ppm in 5-10 minutes, where thesehigh peroxide concentrations were expected to rapidly inactivate theperhydrolytic enzyme catalyst.

When compared to the concentrations of peracids produced by theenzymatic perhydrolysis of esters previously reported, the combinationof the present enzymes and enzyme substrates in the concentration rangesreported herein unexpectedly and efficiently produced an aqueoussolution of peracid at a sufficiently-high concentration to act as abiocidal and virucidal disinfectant, as well as a bleaching agent.Concentrations of peracids in excess of 10 ppm (generally greater thanabout 75 ppm) were produced as described herein using enzyme catalysts(e.g., lipases, proteases), where the resulting peracid-containingsolution, or a dilution thereof, can effect a 5-log or 6-log reductionin infectious bacteria in 5 minutes to 10 minutes at about 25° C.

PCT Publication No. WO2004039418 A1 and U.S. Pat. No. 6,613,505 B2describe a method and composition for destruction of infectious prionproteins in tissue by thermal/enzymatic treatment of the tissue with aprion-destructive protease. U.S. Patent Appl. Pub. No. 2006/0127390 A1describes a method for inactivating agents that cause transmissiblespongiform encephalopathies (also called prion diseases), where thetransmissible spongiform encephalopathy agent is contacted with asubtilisin protease. U.S. Patent Appl. Pub. No. 2006/0030505 A1describes a method for inactivating prions using a composition having aperacid alone, or a peracid and a surfactant; these compositions mayoptionally include one or more enzymes, where the enzymes providedesirable activity for removal of protein-based, carbohydrate-based, ortriglyceride-based soil from substrates. Co-filed provisional patentapplication (Attorney docket number CL3735, titled LOW TEMPERATURE PRIONDECONTAMINATION), filed on Oct. 27, 2006, describes the use of a mixtureof Alcalase and Neutrase in combination with peracid to inactivateprions, herein specifically incorporated by reference.

In another aspect of the invention, cleaning and disinfectingcompositions are provided comprising prion-degrading proteases incombination with in situ generated peracid concentrations havingbiocidal and/or virucidal activity. In yet another aspect, theprion-degrading proteases (e.g., Proteinase K, Pronase, Alcalase® (asubtilisin protease), and Neutrase® (a metallo-endopeptidase, also knownas a neutral protease), are capable of catalyzing the perhydrolysis ofesters and/or amides to produce the corresponding peracid atconcentrations efficacious for disinfection, thereby creating a biocideand/or virucide having prion-degrading activity. Savinase®, Polarzyme®,Everlase® and combinations thereof) are also capable of catalyzing theperhydrolysis of esters and/or amides to produce the correspondingperacid at concentrations efficacious for disinfection, thereby creatinga biocide and/or virucide that may also have prion-degrading activity.In a further aspect of the invention, prion-degrading proteases may beused in combination with an added enzyme having perhydrolytic activity,such as a lipase, at a pH where the protease(s) alone may not produce anefficacious concentration of peracid (e.g., at pH of from 4.0 to 6.5).This combination may also yield a synergistic concentration of peracid,where the peracid is produced in a concentration that is greater thanthe sum of peracid produced by either protease or lipase alone.

Unless otherwise stated, all references cited are hereby specificallyincorporated by reference. Further, when an amount, concentration, orother value or parameter is given either as a range, preferred range, ora list of upper preferable values and lower preferable values, this isto be understood as specifically disclosing all ranges formed from anypair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, preferably with 5% of the reported numerical value.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

As used herein, the term “peracid” is synonymous with peroxyacid, peroxyacid, percarboxylic acid and peroxoic acid. As is commonly known,peracid includes peracetic acid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0.

The term “triacetin” is synonymous with glycerin triacetate; glyceroltriacetate; glyceryl triacetate, 1,2,3-triacetoxypropane,1,2,3-propanetriol triacetate and all other synonyms of CAS RegistryNumber 102-76-1.

As used herein, the terms “suitable enzymatic reaction mixture”,“components suitable for in situ generation of a peracid”, and “suitablereaction components” refer to the materials and water in which thereactants and enzyme catalyst come into contact. The components of thesuitable aqueous reaction mixture are provided herein and those skilledin the art appreciate the range of component variations suitable forthis process. In one embodiment, the suitable enzymatic reaction mixtureproduces peracid in situ upon combining the reaction components. Assuch, the reaction components may be provided as a multicomponent systemwherein one or more of the reaction components remains separated untiluse. The design of systems for combining multiple active components areknown in the art and generally will depend upon the physical form of theindividual reaction components. For example, multiple active fluids(liquid-liquid) systems typically use multichamber dispenser bottles ortwo-phase systems (U.S. Patent Appln. Pub. No. 2005/0139608; U.S. Pat.No. 5,398,846; U.S. Pat. No. 5,624,634; U.S. Pat. No. 6,391,840; E.P.Patent No. 0807156B1; U.S. Patent Appln. Pub. No. 2005/0008526; and PCTPublication No. WO 00/11713A1) such as found in some bleachingapplications wherein the desired bleaching agent is produced upon mixingthe reactive fluids. Other forms of multicomponent systems used togenerate peracid may include, but are not limited to those designed forone or more solid components or combinations of solid-liquid components,such as powders (e.g., many commercially available bleachingcomposition, U.S. Pat. No. 5,116,575), multi-layered tablets (U.S. Pat.No. 6,210,639), water dissolvable packets having multiple compartments(U.S. Pat. No. 6,995,125) and solid agglomerates that react upon theaddition of water (U.S. Pat. No. 6,319,888).

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate with peroxide to form a peracid. Typically, aninorganic peroxide is reacted with the selected substrate in thepresence of a catalyst to produce the peracid. As used herein, the term“chemical perhydrolysis” includes perhydrolysis reactions in which asubstrate (a peracid precursor) is combined with a source of hydrogenperoxide wherein peracid is formed in the absence of an enzyme catalyst.As used herein, the term “enzymatic perhydrolysis” refers to aperhydrolysis reaction that is assisted or catalyzed by an enzymegenerally classified as a hydrolase.

“Carboxylic ester hydrolase” refers to an enzyme that catalyzes thehydrolysis of an ester (E.C. 3.1.1.-). The carboxylic ester hydrolasefamily includes, but is not limited to lipases (e.g., triacylglycerollipases [E.C. 3.1.1.3]) and esterases.

“Lipase” refers to an enzyme that catalyzes the hydrolysis of fats intoglycerol and fatty acids by hydrolyzing ester bonds (EC 3.1.1.3). Somelipases have been reported to have perhydrolysis activity.

“Esterase” refers to an enzyme that catalyzes the hydrolysis of an ester(EC 3.1.1.-). Some esterases have been reported to have perhydrolysisactivity

“Protease” refers to an enzyme that catalyzes the hydrolytic breakdownof proteins via hydrolysis of peptide bonds (EC 3.4.-). As describedherein, some proteases exhibit perhydrolysis activity.

As used herein, the terms “perhydrolase catalyst” and “at least onesuitable enzyme catalyst having perhydrolase activity” refer herein toan enzyme catalyst that is characterized by perhydrolase activity. Theenzyme catalyst is selected from the group consisting of lipases,esterases, proteases, and/or mixtures thereof wherein the catalyst hasperhydrolysis activity. The enzyme catalyst may be in the form of awhole microbial cell, permeabilized microbial cell(s), one or more cellcomponents of a microbial cell extract, partially purified enzyme, orpurified enzyme. As used herein, the term “produced from an organism” isused to describe the source of the suitable catalyst. The enzymecatalyst may be produced from the target organism or may berecombinantly produced in a suitable production host.

As used herein, “one unit of enzyme activity” or “one unit of activity”or “U” is defined as the amount of enzyme activity required for theproduction of 1 μmol of peracid product per minute at a specifiedtemperature.

As used herein, the term “perhydrolase activity” refers to the enzymeactivity per unit mass (for example, milligram) of protein, solid orliquid enzyme-containing composition, dry cell weight, or immobilizedcatalyst weight. Comparisons of perhydrolase activity of catalysts weredetermined proportional to the dry cell weight, solid or liquidenzyme-containing composition or protein catalyst weight.

As used herein, the term “disinfect” refers to the process of cleansingso as to destroy or prevent the growth of pathogenic microorganisms. Asused herein, the term “disinfectant” refers to an agent that disinfectsby destroying, neutralizing, or inhibiting the growth ofdisease-carrying microorganisms. Typically disinfectants are used totreat inanimate objects or surfaces. As used herein, the term“antiseptic” refers to a chemical agent that inhibits the growth ofdisease-carrying microorganisms.

As used herein, the term “virucide” refers to an agent that inhibits ordestroys viruses. An agent that exhibits the ability to inhibit ordestroy viruses is described as having “virucidal” activity. Peracidscan have virucidal activity. Typical alternative virucides known in theart which may be suitable for use with the present invention include,for example, alcohols, ethers, chloroform, formaldehyde, phenols, betapropiolactone, iodine, chlorine, mercury salts, hydroxylamine, ethyleneoxide, ethylene glycol, quaternary ammonium compounds, enzymes, anddetergents

As used herein, the term “biocide” refers to a chemical agent, typicallybroad spectrum, which inactivates or destroys microorganisms. A chemicalagent that exhibits the ability to inactivate or destroy microorganismsis described as having “biocidal” activity. Peracids can have biocidalactivity. Typical alternative biocides known in the art, which may besuitable for use in the present invention include, for example,chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone,acrolein, amines, chlorinated phenolics, copper salts, organo-sulphurcompounds, and quaternary ammonium salts.

As used herein, the phrase “minimum biocidal concentration” refers tothe minimum concentration of a biocidal agent that, for a specificcontact time, will produce a desired lethal, irreversible reduction inthe viable population of the targeted microorganisms. The effectivenesscan be measured by the log₁₀ reduction in viable microorganisms aftertreatment. In one aspect, the targeted reduction in viable cells aftertreatment is a 3-log reduction, more preferably a 4-log reduction, andmost preferably at least a 5-log reduction. In another aspect, theminimum biocidal concentration is a 6-log reduction in viable microbialcells.

As used herein, the terms “prion”, “prion particle”, and “infectionprion particle” refer to infectious proteins associated withneurodegenerative diseases including, but not limited to scrapie, bovinespongiform encephalopathy (BSE), trasmissible spongiform encephalopathy(TSE), chronic wasting disease, and Creutzfeldt-Jacob disease. The term“prion-destructive protease” or “prion-degrading protease” refers to atleast one protease (preferably combinations of two or more proteases)useful for degrading or destroying infectious prion particles (forexample, see WO 2004039418 A1 and US 20020172989 A1). In one embodiment,the prion-destructive protease is selected from Proteinase K, Pronase,and mixtures thereof. In a preferred embodiment, the prion-destructiveprotease is a mixture of Proteinase K and Pronase®. In a furtherpreferred embodiment, the prion-destructive protease is selected fromAlcalase®, Neutrase®, and mixtures thereof. In an additional preferredembodiment, Savinase®, Polarzyme®, and Everlase®, and mixtures thereof,are employed as perhydrolytic enzymes, where these proteases,individually, or in combination, may also degrade prions. In yet afurther preferred embodiment, the prion-destructive protease is amixture of Alcalase® and Neutrase®.

In one aspect, the peracids formed by the present process can be used toreduce a microbial population when applied on and/or at a locus. As usedherein, a “locus” of the invention comprises part or all of a targetsurface suitable for disinfecting or bleaching. Target surfaces includeall surfaces that can potentially be contaminated with microorganisms,viruses, prions or combinations thereof. Non-limiting examples includeequipment surfaces found in the food or beverage industry (such astanks, conveyors, floors, drains, coolers, freezers, equipment surfaces,walls, valves, belts, pipes, drains, joints, crevasses, combinationsthereof, and the like); building surfaces (such as walls, floors andwindows); non-food-industry related pipes and drains, including watertreatment facilities, pools and spas, and fermentation tanks; hospitalor veterinary surfaces (such as walls, floors, beds, equipment, (such asendoscopes) clothing worn in hospital/veterinary or other healthcaresettings, including scrubs, shoes, and other hospital or veterinarysurfaces); restaurant surfaces; bathroom surfaces; toilets; clothes andshoes; surfaces of barns or stables for livestock, such as poultry,cattle, dairy cows, goats, horses and pigs; and hatcheries for poultryor for shrimp. Additional surfaces also include food products, such asbeef, poultry, pork, vegetables, fruits, seafood, combinations thereof,and the like. The locus can also include water absorbent materials suchas infected linens or other textiles. The locus also includes harvestedplants or plant products including seeds, corms, tubers, fruit, andvegetables, growing plants, and especially crop growing plants,including cereals, leaf vegetables and salad crops, root vegetables,legumes, berried fruits, citrus fruits and hard fruits.

Non-limiting examples of surface materials are metals (e.g., steel,stainless steel, chrome, titanium, iron, copper, brass, aluminum, andalloys thereof), minerals (e.g., concrete), polymers and plastics (e.g.,polyolefins, such as polyethylene, polypropylene, polystyrene,poly(meth)acrylate, polyacrylonitrile, polybutadiene,poly(acrylonitrile, butadiene, styrene), poly(acrylonitrile, butadiene),acrylonitrile butadiene; polyesters such as polyethylene terephthalate;and polyamides such as nylon). Additional surfaces include brick, tile,ceramic, porcelain, wood, vinyl, linoleum, and carpet.

Suitable Aqueous Reaction Conditions for the Enzyme-CatalyzedPreparation of Peracids from Carboxylic Acid Esters and/or Amides andHydrogen Peroxide

A process is provided to produce an aqueous mixture comprising a peracidby reacting a suitable substrate, at least one enzyme catalyst havingperhydrolysis activity, and a source of peroxygen providing a hydrogenperoxide concentration of 500 mM or more at a neutral to acidic pH. Asused herein, the terms “peroxygen source” and “source of peroxygen”refer to compounds capable of providing hydrogen peroxide at aconcentration of about 500 mM or more when in an aqueous solutionincluding, but not limited to hydrogen peroxide, hydrogen peroxideadducts, perborates, and percarbonates. As described herein, theperoxygen source is capable of providing, upon combining the reactioncomponents, a mixture having a hydrogen peroxide concentration of atleast 500 mM.

As used herein, the terms “suitable substrate”, “peracid precursor”, and“bleach activator” will be used to describe substrates capable ofundergoing enzymatic perhydrolysis to generate a peracid using thepresent reaction conditions. The substrates may also undergo partialchemical perhydrolysis in the presence or absence of enzyme. As such,substrates suitable in the present invention are those that can undergoenzymatic perhydrolysis to generate at least 10 ppm more peracid thenthat produced chemically under identical reaction conditions, preferablyat least 100 ppm, more preferably at least 250 ppm, even more preferablyat least 500 ppm, yet even more preferably at least 1000 ppm, still evenmore preferably at least 2000 ppm, and most preferably at least 5000 ppmperacid.

Suitable substrates may include one or more carboxylic acid esters,amides, glycerides (mono-, di-, and/or triglycerides) or mixture thereofcapable of undergoing enzymatic perhydrolysis under the present reactionconditions. In one embodiment, the substrate may be optionallysubstituted, especially with alkoxy and/or hydroxyl groups. In anotherembodiment, the substrate may be a acylated polyol. In yet anotherembodiment, the acylated polyol is selected from the group consisting ofmono-, di- or polyacylated polyols derived from glycerol, erythritol,threitol, xylitol, ribitol, arabitol, mannitol, and sorbitol.

In a preferred embodiment, suitable substrates include carboxylic acidesters, amides, and/or glycerides having a formula selected from thegroup consisting of:

a) esters of the formula

wherein R₁=C1 to C10 strain chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₂=C1 to C10strain chain or branched chain alkyl group, (CH₂CH₂—O)_(n)H or(CH₂CH(CH₃)—O)_(n)H and n=1 to 10;b) glycerides of the formula

wherein R₁=C1 to C10 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); andc) amides of the formula

wherein R₅ and R₆=H or a C1 to C5 straight chain or branched alkylgroup. In one embodiment, the substrate may be a mixture of one or moresuitable substrates as described herein.

In a preferred embodiment, the substrates are selected from the groupconsisting of methyl lactate, ethyl lactate, methyl glycolate, ethylglycolate, methyl methoxyacetate, ethyl methoxyacetate, methyl3-hydroxybutyrate, ethyl 3-hydroxybutyrate, triethyl 2-acetyl citrate,glucose pentaacetate, gluconolactone, monoacetin, diacetin, triacetin,monobutyrin, dibutyrin, tributyrin, glyceryl monooctanoate, glyceryldioctanoate, glyceryl trioctanoate, acetamide, diacetamide, and mixturesthereof.

In yet a further preferred embodiment, the substrate is selected fromthe group consisting of monoacetin, diacetin, triacetin, acetamide,diacetamide, and mixtures thereof.

The enzyme substrate is present in the reaction mixture at aconcentration sufficient to produce the desired concentration of peracidupon enzyme-catalyzed perhydrolysis. It has been demonstrated in theaccompanying examples that there are preferred combinations of enzymesubstrate and enzyme catalyst that produce a desirable concentration ofperacid. The substrate need not be completely soluble in the reactionmixture, but have sufficient solubility to permit conversion of theester or amide by the enzyme catalyst to the corresponding peracid. Thesubstrate is present in the reaction mixture at a concentration of 0.05wt % to 40 wt % of the reaction mixture, preferably at a concentrationof 0.1 wt % to 20 wt % of the reaction mixture, and more preferably at aconcentration of 0.5 wt % to 10 wt % of the reaction mixture. Preferredsubstrates when using a lipase, esterase, or protease as catalyst, orcombinations of lipases and/or proteases as catalyst, includemonoacetin, diacetin, triacetin, and mixtures ofmonoacetin/diacetin/triacetin. Additional preferred substrates whenusing a protease as catalyst, or combinations of lipases and proteasesas catalyst, include acetamide, diacetamide, and mixtures thereof.

The peroxygen source may include, but is not limited to, hydrogenperoxide, perborate salts (e.g., sodium perborate) and percarbonatesalts (e.g. sodium percarbonate). The concentration of peroxygencompound in the reaction mixture may range from 0.1 wt % to about 50 wt%, preferably from 1 wt % to about 40 wt %, more preferably from 2 wt %to about 30 wt %.

The concentration of the hydrogen peroxide provided by the peroxygencompound in the aqueous reaction mixture is initially at least 500 mM ormore upon combining the reaction components. In one embodiment, thehydrogen peroxide concentration in the aqueous reaction mixture is 1000mM or more. In another embodiment, the hydrogen peroxide concentrationin the aqueous reaction mixture is 2500 mM or more.

The molar ratio of the hydrogen peroxide to substrate (H₂O₂:substrate)in the aqueous reaction mixture may be from about 0.1 to 20, preferablyabout 0.5 to 10, and most preferably about 2 to 5.

The reaction components may be combined in individual batches or may becombined using a continuous process.

The enzyme catalyst is chosen from the class of hydrolytic enzymes thatincludes esterases, lipases and proteases (EC 3.1.1.-, EC 3.1.1.3, andEC 3.4.-.-, respectively). In particular, the enzymes that are useful inthe present invention are hydrolytic enzymes, such as esterases,lipases, and proteases, whose catalytic activity is normally thehydrolysis of an ester to the corresponding carboxylic acid and alcohol,or the hydrolysis of an amide to the corresponding carboxylic acid andammonia or amine, where in the presence of hydrogen peroxide (or afunctionally-equivalent peroxygen containing compound) the ester oramide is subject to an enzyme-catalyzed perhydrolysis, producing thecorresponding percarboxylic acid.

In one aspect, the enzyme catalyst is a lipase derived from a eukaryoticor prokaryotic organism. In another aspect, the lipase is derived froman organism selected from the genera consisting of Aspergillus,Rhizopus, Penicillium, Candida, Pseudomonas, Mucor, Thermomyces,Alcaligenes, and Sus. In a preferred aspect, the source of the lipase isselected from the group consisting of Aspergillus niger, Rhizopusoryzae, Penicillium sp. I, Penicillium sp. II, Candida rugosa, Candidaantartica lipase A, Candida antartica lipase B, Pseudomonas cepacia,Pseudomonas fluorescens, Thermomyces languinosus, Mucor miehei, Susscrofa, and Alcaligenes sp. In a further preferred aspect, the lipase isselected from the group consisting of Aspergillus sp. lipase,Aspergillus niger lipase, Candida antartica lipase B, Pseudomonas sp.lipase, Alcaligenes sp. lipase, Candida rugosa lipase, Rhizopus oryzaelipase, and mixtures thereof. Many of the lipases of exemplified hereinwere obtained from BioCatalytics Inc. (Pasadena, Calif.) and referred toby their corresponding catalog number (Example 1, Table 1). Accordingly,in a further preferred aspect, the lipase is selected from the groupconsisting of ICR-101 Aspergillus sp. lipase, ICR-102 Rhizopus sp.lipase, ICR-103 Rhizopus oryzae lipase, ICR-104 Penicillium sp. Ilipase, ICR-105 Penicillium sp. II lipase, ICR-106 Candida rugosalipase, ICR-107 Pseudomonas cepacia lipase, ICR-108 Pseudomonas sp.lipase, ICR-109 Pseudomonas fluorescens lipase, ICR-110 Candidaantartica lipase B, ICR-111 Candida sp. lipase, ICR-112 Candidaantartica lipase A, ICR-113 Pseudomonas sp. lipase, ICR-114 porcinepancreas lipase, ICR-115 Thermomyces languinosus lipase, ICR-116 Mucormiehei lipase, and ICR-117 Alcaligenes sp. lipase. In a furtherpreferred aspect, the lipase is selected from the group consisting ofLipase AY “Amano” 30 (Candida rugosa lipase), Lipase R “Amano”(Penicillium roqueforti lipase), Lipase F-AP15 (Rhizopus oryzae lipase),Lipase M “Amano” 10 (Mucor javanicus lipase), Lipase A “Amano” 12(Aspergillus niger lipase), Lipase G “Amano” 50 (Penicillium camembertiilipase), Amano F-DS lipase (Rhizopus oryzae lipase), Amano DS lipase(Aspergillus niger lipase) (all from Amano), and CALB L (liquidformulation of Candida antartica lipase B), Novozym 435 (immobilizedCandida antartica lipase B), Lipozyme TL (Thermomyces lanuginosuslipase), Palatase 20000L (Aspergillus oryzae lipase) (all fromNovozymes), and Validase AN lipase (Aspergillus niger lipase), DietrenzCR lipase (Candida rugosa lipase) (all from Valley Research), and EnzecoMLC lipase (a microbial lipase concentrate from Aspergillus niger soldby Enzyme Development Corporation. Most preferably, the lipase isselected form the group consisting of BioCatalytics ICR-101 (Aspergillussp. lipase), BioCatalytics ICR-110 (Candida antartica lipase B),BioCatalytics Chirazyme L2 lipase (C. antartica lipase B), BioCatalyticsICR-113 (Pseudomonas sp. lipase), BioCatalytics CR-117 (Alcaligenes sp.lipase), Lipase A “Amano” 12 (Aspergillus niger lipase), Novozym CALB L(Candida antartica lipase B), Novozym 435 (immobilized Candida antarticalipase B), Novozym Palatase 20000L (Aspergillus oryzae lipase), andValley Research Validase AN (Aspergillus niger lipase).

In one embodiment, the enzyme catalyst is a protease (E.C. 3.4.-.-)derived from a eukaryotic or prokaryotic organism. In another embodimentthe protease is derived from an organism selected from the generaconsisting of Aspergillus, Streptomyces, Tritirachium, Rhizopus,Bacillus, Sus, Carica, Ananas, Pseudomonas, Mucor, Thermomyces,Alcaligenes, and Sus. In a preferred aspect, the source of the proteaseis selected from the group consisting of Aspergillus saitoi, Rhizopussp, Bacillus sp., Bacillus subtilis, Sus scrofa (pig), Carica papaya(papaya), Ananas comosus (pineapple), Streptomyces griseus, andTritirachium album. In a preferred embodiment, the protease is selectedfrom the group consisting of the Aspergillus saitoi type XIII protease,Rhizopus sp. type XVIII protease, Bacillus sp. (e.g. Bacillus subtilis,Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus lentus,etc.) proteases such as subtilisin (Alcalase®; available fromNovozymes), the neutral protease (Neutrase®; available from Novozymes),(Savinase®; available from Novozymes), Sus scrofa pepsin, Carica papayaChymopapain, Ananas comosus bromelain, Carica papaya papain,Streptomyces griseus Pronase (also known as Pronase E® or Pronase®),Trititrachium album Proteinase K (including recombinantly producedProteinase K from Pichia pastoris), and mixtures thereof.

Many enzyme catalysts (whole cell, partially purified, or purified) havebeen reported to have catalase activity (EC 1.11.1.6). Catalasescatalyze the conversion of hydrogen peroxide into oxygen and water. In apreferred embodiment, the enzyme catalyst lacks significant catalaseactivity or is engineered or purified to decrease or eliminate catalaseactivity.

The concentration of enzyme catalyst employed in the aqueous reactionmixture depends in part on the specific catalytic activity of the enzymecatalyst, and is chosen to obtain the desired rate of reaction. Theweight of soluble enzyme used as catalyst in perhydrolysis reactionstypically ranges from 0.01 mg to 10 mg of enzyme per mL of totalreaction volume, preferably from 0.10 mg to 2.0 mg of enzyme per mL. Theenzyme may also be immobilized on a soluble or insoluble support usingmethods well-known to those skilled in the art; see for example,Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor;Humana Press, Totowa, N.J., USA; 1997. The use of immobilized enzymespermits the recovery and reuse of the catalyst in subsequent reactions.Additional forms of the enzyme catalyst that are useful in the presentapplication include whole microbial cells, cell extracts, andpartially-purified enzymes. These additional forms of the catalyst mayalso be immobilized using the methods referenced above.

In one aspect, the concentration of peracid generated by the combinationof chemical perhydrolysis and enzymatic perhydrolysis of the substrateis sufficient to provide an effective concentration of peracid forbleaching or disinfection at a desired pH. In another aspect, thepresent methods provide combinations of enzymes and enzyme substrates toproduce the desired effective concentration of peracid, where, in theabsence of added enzyme, there is a significantly lower concentration ofperacid produced. Although there may in some cases be substantialchemical perhydrolysis of the enzyme substrate by direct chemicalreaction of inorganic peroxide with the enzyme substrate, there may notbe a sufficient concentration of peracid generated to provide aneffective concentration of peracid in the desired applications, and asignificant increase in total peracid concentration is achieved by theaddition of an appropriate enzyme catalyst to the reaction mixture.

The present process produces a concentrated aqueous peracid solutionthat may be optionally diluted (e.g., with water) prior to use. As usedherein, “peracid solution”, “aqueous peracid solution”, and“concentrated aqueous peracid solution” will refer to the concentratedperacid solution generated by the present enzyme-catalyzed perhydrolysisprocess. In one embodiment, the “concentrated aqueous peracid solution”comprises at least 10 ppm peracid, preferably at least 100 ppm peracid,more preferably at least 200 ppm peracid, even more preferably at least250 ppm peracid, yet even more preferably at least 500 ppm, still yeteven more preferably at least 1000 ppm, yet even more preferably atleast 2000 ppm, and most preferably at least 5000 ppm. The productmixture comprising the peracid may be optionally diluted with water, ora solution predominantly comprised of water, to produce a mixture withthe desired lower concentration of peracid. The decision to dilute theaqueous peracid solution produced by the present method will depend upona variety of factors including, but not limited to the targetapplication (animal health disinfectant, instrument sterilization,household cleaner, bleaching agent, etc.), temperature and time requiredto ensure the desired level efficacy for the target application, theamount of soil at and/or on the target locus, and the target pathogen'ssusceptibility to peracid disinfectants. The reaction time required toproduce the desired concentration of peracid is not greater than abouttwo hours, preferably not greater than about 30 minutes, more preferablynot greater than about 10 minutes, and most preferably less than orabout 5 minutes. As the reaction continues, the concentration of peracidwill increase to the point of equilibrium. As such, the presentinvention is directed to a process that produces a high concentrationwithin about 5 minutes, said concentration may continue to rise until anequilibrium of the reaction is reached.

The temperature of the peracid synthesis reaction is chosen to controlboth the reaction rate and the stability of the enzyme catalystactivity. The temperature of the reaction may range from just above thefreezing point of the reaction mixture (approximately 0° C.) to about65° C., with a preferred range of reaction temperature of from about 5°C. to about 35° C.

The pH of the final reaction mixture (i.e., pH of the reaction mixtureupon combining the reaction components) containing peracid is 2.5 to7.5, preferably from 3 to 7, more preferably from 3.5 to 6.5, and mostpreferably 4 to 6.5. The pH of the reaction, and of the final reactionmixture, may optionally be controlled by the addition of a suitablebuffer, including, but not limited to phosphate, pyrophosphate,bicarbonate, acetate, or citrate. The concentration of buffer is from0.1 mM to 1.0 M, preferably from 1 mM to 100 mM, most preferably from 10mM to 50 mM.

In another aspect, the enzymatic perhydrolysis product may containadditional components that provide desirable functionality. Theseadditional components include, but are not limited to detergentbuilders, emulsifiers, surfactants, corrosion inhibitors, enzymestabilizers, and peroxide stabilizers (e.g., metal ion chelatingagents). Many of the additional components are well know in thedetergent industry (see for example U.S. Pat. No. 5,932,532 herebyincorporated by reference). Examples of emulsifiers include polyvinylalcohol or polyvinylpyrrolidine. Examples of surfactants, including a)non-ionic surfactants such as block copolymers of ethylene oxide orpropylene oxide, ethoxylated or propoxylated linear and branched primaryand secondary alcohols, and aliphatic phosphine oxides b) cationicsurfactants such as such as quaternary ammonium compounds, particularlyquaternary ammonium compounds having a C8-C20 alkyl group bound to anitrogen atom additionally bound to three C1-C2 alkyl groups, c) anionicsurfactants such as alkane carboxylic acids (e.g., C8-C20 fatty acids),alkyl phosphonates, alkane sulfonates (e.g., sodium dodecylsulphate“SDS”) or linear or branched alkyl benzene sulfonates, alkene sulfonatesand d) amphoteric and zwitterionic surfactants such as aminocarboxylicacids, aminodicarboxylic acids, and alkybetaines. Additional componentsmay include fragrances, dyes, stabilizers of hydrogen peroxide (e.g.,1-hydroxyethylidene-1,1-diphosphonic acid (Dequest 2010, Solutia Inc.,St. Louis, Mo.)), stabilizers of enzyme activity (e.g.,polyethyleneglycol (PEG)), detergent builders and metal chelators (e.g.,ethylenediaminetetraacetic acid (EDTA)).

Enzymatic In Situ Production of Peracids

The present aqueous enzymatic method produces high concentrations ofperacid or peracids in situ. The peracids produced are quite reactiveand relatively unstable, generally decreasing in concentration overtime. As such, it may be desirable to keep the various reactioncomponents separated, especially for liquid formulations. In one aspect,the hydrogen peroxide source is separate from either the substrate orthe enzyme, preferably from both. This can be accomplished using avariety of techniques including, but not limited to the use ofmulticompartment chambered dispensers (U.S. Pat. No. 4,585,150) andphysically combining the enzyme catalyst with the present substrates andhydrogen peroxide source to initiate the aqueous enzymatic perhydrolysisreaction. The enzyme catalyst may be immobilized within the body of thereaction chamber or separated (e.g. filtered, etc.) from the reactionproduct comprising the peracid prior to contacting the surface and/orobject targeted for treatment. The enzyme catalyst may be in a liquidmatrix or in a solid form (i.e. powdered, tablet) or embedded within asolid matrix that is subsequently mixed with the substrates and hydrogenperoxide source to initiate the enzymatic perhydrolysis reaction. In afurther aspect, the enzyme catalyst may be contained within adissolvable or porous pouch that may be added to the aqueous substratematrix to initiate enzymatic perhydrolysis.

HPLC Assay Method for Determining the Concentration of Peracid andHydrogen Peroxide.

A variety of analytical methods can be used in the present method toanalyze the reactants and products including, but not limited totitration, high performance liquid chromatography (HPLC), gaschromatography (GC), mass spectroscopy (MS), and capillaryelectrophoresis (CE).

The analytical procedure described by U. Karst et al. (Anal. Chem., 69(17):3623-3627 (1997)) was employed, as described herein, for analysisof product mixtures containing peracid and hydrogen peroxide. Briefly,the concentration of peracetic acid (PAA) in analyzed samples rangedfrom 0.025 mM-10 mM, and the concentration of H₂O₂ ranged from 0.075mM-3 mM. Reaction mixtures containing peracid and/or hydrogen peroxidewere, if necessary prior to analysis, diluted to produce a concentrationof peracid or peroxide in these ranges. Into a 4-mL vial was placed0.100 mL of 20 mM methyl p-tolyl sulfide (MTS) in acetonitrile, 0.300 mLof distilled and deionized water (dd) and 0.100 mL of sample solution(undiluted or diluted with dd water by a factor of up to 1:25 foranalysis of peracid), or 0.100 mL of 20 mM MTS in acetonitrile and 0.390mL of dd water were added to 0.010 mL of a 1:10 dilution of samplesolution (for analysis of hydrogen peroxide). After a reaction time of10 minutes (in the dark, with no stirring), 0.400 mL CH₃CN and 0.100 mLof 40-mM triphenylphosphine (TPP) in CH₃CN were added to start thesecond derivatization reaction for detection of peroxide. The solutionwas left standing in the dark for 30 min to complete the assay reaction.At the end of 30 minutes, 0.100 mL of 10 mM N,N-diethyl-m-toluamide(DEET, HPLC external standard) was added and the resulting solutionanalyzed by HPLC: Supelco Discovery C8 10-cm column with pre-column,10-μL injection, UV detection at 225 nm, solvent A: acetonitrile,solvent B: deionized water, 1 mL/min gradient as follows:

Time (min:sec) % CH₃CN % H₂O 0:00 40 60 3:00 40 60 3:10 100 0 4:00 100 04:10 40 60 7:00 (stop) 40 60

Determination of Minimum Biocidal Concentration of Peracids

The method described by J. Gabrielson, et al. (J. Microbiol. Methods 50:63-73 (2002)) was employed for determination of the Minimum BiocidalConcentration (MBC) of peracids, or for determination of the MBC ofhydrogen peroxide and enzyme substrates. The assay method is based onXTT reduction inhibition, where XTT((2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium,inner salt, monosodium salt) is a redox dye that indicates microbialrespiratory activity by a change in optical density (OD) measured at 490nm or 450 nm. However, there are a variety of other methods availablefor testing the activity of disinfectants and antiseptics including, butnot limited to viable plate counts, direct microscopic counts, dryweight, turbidity measurements, absorbance, and bioluminescence (see,for example Brock, Semour S., Disinfection, Sterilization, andPreservation, 5^(th) edition, Lippincott Williams & Wilkins,Philadelphia, Pa., USA; 2001).

Determination of Virucidal Activity for Disinfectants and Antiseptics

Methods to evaluate the virucidal activity of disinfectants andantiseptics are well known in the art (Brock, S., supra and Papageorgiouet al., Appl. Environ. Microbiol., 67(12):5844-5848 (2001)). The presentperacid compositions are expected to exhibit virucidal activity. It hasbeen reported that acidic conditions (pH <7) may enhance virucidalactivity. Accordingly, in a further aspect, the present peracidcomposition may be comprised of an acidic pH (pH <7). In anotherembodiment, the pH is less than 6.5, preferably less than 6, morepreferably less than about 5.

Uses of Enzymatically Prepared Peracid Compositions

The enzyme-generated peracid produced according to the present methodscan be used in a variety of applications for reduction of microbial,fungal, viral, and infectious protein (i.e., prion) contamination at alocus as defined herein, such as for the decontamination of medicalinstruments (e.g., endoscopes), textiles (e.g., garments, carpets), foodpreparation surfaces, food storage and food-packaging equipment,materials used for the packaging of food products, chicken hatcheriesand grow-out facilities, animal enclosures, water treatment facilities,pools and spas, fermentation tanks, and spent process waters that havemicrobial and/or virucidal activity. In a preferred aspect, the presentperacid compositions are particularly useful as a cleaning anddisinfecting agent for non-autoclavable medical instruments and foodpackaging equipment. As the peracid-containing formulation may beprepared using GRAS or food-grade components (enzyme, enzyme substrate,hydrogen peroxide, and buffer), the enzyme-generated peracid may also beused for decontamination of animal carcasses, meat, fruits andvegetables, or for decontamination of prepared foods. Theenzyme-generated peracid may be incorporated into a product whose finalform is a powder, liquid, gel, solid or aerosol. The enzyme-generatedperacid may be diluted to a concentration that still provides anefficacious decontamination.

The compositions comprising an efficacious concentration of peracid canbe used to clean and disinfect surfaces and/or objects contaminated (orsuspected of being contaminated) with pathogenic microorganisms,viruses, and/or prions by contacting the surface or object with theproducts produced by the present processes. As used herein, “contacting”refers to placing a disinfecting composition comprising an effectiveconcentration of peracid in contact with a locus suspected ofcontamination with a disease-causing entity for a period of timesufficient to clean and disinfect. The time, temperature, and effectiveconcentration used when contacting the desired locus can be easilydetermined by one of skill in the art. Contacting includes spraying,treating, immersing, flushing, pouring on or in, mixing, combining,painting, coating, applying, affixing to and otherwise communicating aperacid solution comprising an efficacious concentration of peracid withthe surface or inanimate object suspected of being contaminated.

Peracid Disinfectants Having Prion-Degrading Activity

Patent applications PCT Publication No. WO 2004039418 A1, an U.S. PatentAppln. Pub. No. 20020172989 A1, and co-filed US provisional patentapplication (D-Gen provisional patent application title) describemethods for the decontamination of prions that employ a protease ormixture of proteases to destroy the infectious prion protein (Langeveldet al., J. Infect. Diseases, 188:1782-1789 (2003)). The present examplesalso demonstrate that proteases capable of decontaminating infectiousprions are capable of catalyzing the perhydrolysis of esters or amidesto produce the corresponding peracid at concentrations efficacious fordisinfection, thereby making possible the production of a mixturecontaining both an antimicrobial peracid and prion-degrading protease(s)using a single enzyme system.

Proteases for Biocidal Compositions Having Enhanced Prion-DegradingActivity

Enzymes cleaving the amide linkages in protein substrates are classifiedas proteases, or (interchangeably) peptidases (see Walsh, EnzymaticReaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3(1979) and Rao et al., Microbiol. Mol. Biol. Rev., 62(3):597-635(1998)). It includes any enzyme belonging to the EC 3.4 enzyme group.These enzymes can be grossly subdivided into two major categories,exopeptidases and endopeptidases, depending upon their site of action.

As proteases are ubiquitous to all living organisms, suitableprion-degrading proteases can be isolated from a variety of eukaryoticand/or prokaryotic organisms. Commercially-useful proteases have beenisolated from plants (e.g. papain, bromelain, and keratinases), animals(e.g., trypsin, pepsin, and rennin), and microbes such as fungi (e.g.,Aspergillus oryzae proteases) and bacteria (especially well-knownproteases isolated from the genus Bacillus). Bacillus species secretetwo extracellular types of protease, neutral proteases (many of whichare metallo-endoproteases, such as Neutrase®, typically active in ageneral pH range of 5 to 8) and alkaline proteases (such as Alcalase®,typically characterized by high activity at alkaline pH).

A sub-group of the serine proteases tentatively designated subtilaseshas been proposed by Siezen et al., supra. They are defined by homologyanalysis of more than 40 amino acid sequences of serine proteasespreviously referred to as subtilisin-like proteases. A subtilisin waspreviously defined as a serine protease produced by Gram-positivebacteria or fungi, and according to Siezen et al. now is a subgroup ofthe subtilases (or “subtilisin proteases”). A wide variety ofsubtilisins have been identified, and the amino acid sequence of anumber of subtilisins have been determined. These include more than sixsubtilisins from Bacillus strains, namely, subtilisin 168, subtilisinBPN′, subtilisin Carlsberg, subtilisin Y, subtilisin amylosacchariticus,and mesentericopeptidase (Kurihara et al., J. Biol. Chem. 247 5629-5631(1972); Wells et al., Nucleic Acids Res. 11 7911-7925 (1983); Stahl andFerrari, J. Bacteriol. 159 811-819 (1984), Jacobs et al., Nucl. AcidsRes. 13 8913-8926 (1985); Nedkov et al., Biol. Chem. Hoppe-Seyler 366421-430 (1985), Svendsen et al., FEBS Lett. 196 228-232 (1986)), onesubtilisin from an actinomycetales, thermitase from Thermoactinomycesvulgaris (Meloun et al., FEBS Lett. 198 195-200 (1985)), and one fungalsubtilisin, Proteinase K from Tritirachium album (Jany and Mayer (1985)Biol. Chem. Hoppe-Seyler 366 584-492 (1985)). For further reference, seeTable I from Siezen et al., supra.

Subtilisins are well-characterized physically and chemically. Inaddition to knowledge of the primary structure (amino acid sequence) ofthese enzymes, over 50 high resolution X-ray structures of subtilisinshave been determined which delineate the binding of substrate,transition state, products, at least three different proteaseinhibitors, and define the structural consequences for natural variation(Kraut, Ann. Rev. Biochem. 46 331-358 (1977)).

One subgroup of the subtilases, I-S1, comprises the “classical”subtilisins, such as subtilisin 168, subtilisin BPN′, subtilisinCarlsberg (e.g., Alcalase®, available from Novozymes A/S, Bagsvaerd,Norway), and subtilisin DY.

A further subgroup of the subtilases I-S2, is recognized by Siezen etal., (supra). Sub-group I-S2 proteases are described as highly alkalinesubtilisins and comprise enzymes such as subtilisin PB92 (e.g.,Maxacal®, Gist-Brocades NV, Denmark), subtilisin 309 (e.g., Savinase®,Novozymes), subtilisin 147 (e.g., Esperase®, Novozymes), and alkalineelastase YaB.

Random and site-directed mutations of the subtilase gene have botharisen from knowledge of the physical and chemical properties of theenzyme and contributed information relating to subtilase's catalyticactivity, substrate specificity, tertiary structure, etc. (Wells et al.,Proc. Natl. Acad. Sci. U.S.A. 84:1219-1223 (1987); Wells et al., Phil.Trans. R. Soc. Lond. A. 317:415-423 (1986); Hwang and Warshel, Biochem.26 2669-2673 (1987); Rao et al., Nature 328:551-554 (1987); Carter etal., Proteins 6:240-248 (1989); Graycar et al., Annals of the New YorkAcademy of Sciences 672 71-79 (1992); and Takagi, Int J. Biochem. 25307-312 (1993).

Examples of proteases and protease variants have been disclosed innumerous United States patents and patent applications including, butnot limited to U.S. patent application publications U.S. 200502391885 A1and U.S. 20060147499 A1 and issued U.S. Pat. No. 5,500,364; U.S. Pat.No. 6,506,589; U.S. Pat. No. 6,555,355; U.S. Pat. No. 6,558,938; U.S.Pat. No. 6,558,939; U.S. Pat. No. 6,605,458; U.S. Pat. No. 6,632,646;U.S. Pat. No. 6,682,924; U.S. Pat. No. 6,773,907; U.S. Pat. No.6,777,218; U.S. Pat. No. 6,780,629; U.S. Pat. No. 6,808,913; U.S. Pat.No. 6,835,821; U.S. Pat. No. 6,893,855; U.S. Pat. No. 6,921,657; U.S.U.S. Pat. No. 7,026,53; U.S. Pat. No. 7,098,017; and U.S. Pat. No.7,109,016 (each hereby incorporated by reference in their entirety).

Examples of well-known, commercially-available proteases include, butare not limited to Pronase® (CAS #9036-06-0; a protease mixture fromStreptomyces griseus available from Calbiochem), Proteinase K (a fungalsubtilisin from Tritirachium album), Alcalase® (Novozymes; also known assubtilisin Carlsberg or subtilisin A from Bacillus licheniformis; seeGenBank® Accession No. P00780), Neutrase® (Novozymes; a neutral proteasefrom Bacillus amyloliquefaciens; available from Sigma-Aldrich, catalog#P1236), Everlase® (Novozymes; a protease from Bacillus sp. availablefrom Sigma-Aldrich, catalog #P5985), Polarzyme® (Novozymes; a proteaseengineered for low temperature washing applications), and Savinase®(Novozymes, also referred to as subtilisin 309 from Bacillus lentus; seeGenBank® Accession No. P29600). The commercially-available enzymes maybe incorporated into the present compositions and methods in a varietyof product forms including powers powders, tablets, and liquidformulations. In another aspect, the commercially-available enzymes(s)may be optionally purified or partially purified prior to use in thepresent formulations. Means to purify proteins are well-known in theart.

Biocidal Compositions Comprising Prion-Degrading Proteases

Biocidal compositions comprising at least one prion-degrading proteaseand components suitable for in situ generation of a peracid are provided(i.e. a multifunctional biocidal composition comprising proteases toenhance prion degradation). In one aspect, the biocidal compositioncomprises a mixture of two or more prion-degrading proteases andcomponents suitable for in situ generation of a peracid. In anotheraspect, one or more of the prion-degrading proteases also providesperhydrolysis activity for generating peracids from the presentsubstrates.

In one embodiment, the present biocidal compositions includes componentsfor in situ production (produced chemically and/or enzymatically) of atleast one peracid. In a preferred embodiment, the peracid is primarilyproduced enzymatically from a suitable substrate as defined herein. Inanother preferred embodiment, the suitable substrate is a glyceridesubstrate selected from the group consisting of monoacetin, diacetin,triacetin, and mixtures thereof. In another preferred aspect, theprion-degrading biocidal composition comprises in situ generatedperacetic acid (and/or reaction components suitable for producingperacetic acid).

In another aspect, the present prion-degrading biocidal compositioncomprises at least one lipase and/or esterase having perhydrolysisactivity for generating at least one peracid from the present substratesand at least one protease having prion-degrading activity. In a highlypreferred aspect, the prion-degrading biocidal compositions comprise twoor more proteases, wherein one or more of the proteases may alsoprovides provide additional perhydrolysis activity.

In a preferred aspect, the source organism of the prion-degradingprotease is selected from the group consisting of Bacillus subtilis,Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus lentus,Streptomyces griseus, and Tritirachium album. In a preferred embodiment,suitable prion-degrading proteases are selected from the groupconsisting of Alcalase® (available from Novozymes), Savinase® (availablefrom Novozymes), Neutrase® (available from Novozymes), Polarzyme®(available from Novozymes), Everlase® (available from Novozymes),Streptomyces griseus Pronase (also known as Pronase E® or Pronase®;available from Calbiochem, La Jolla, Calif.), Tritirachium albumProteinase K (including recombinantly produced Proteinase K from Pichiapastoris), and mixtures thereof.

Combinations of several proteases have been identified as beingparticularly effective for enhancing prion degradation. In a preferredaspect, the biocidal composition comprises a mixture of Pronase® andProteinase K and components suitable for in situ generation of aperacid. In another preferred aspect, a biocidal composition is providedcomprising a mixture of Alcalase® and Neutrase® and components suitablefor in situ generation of a peracid. In a further aspect, the biocidalcomposition comprises the above prion-degrading proteases and in situgenerated peracid. In a preferred aspect, the in situ generated peracidis peracetic acid.

This composition comprising prion-degrading proteases and an efficaciousconcentration of a peracid may optionally include one or moresurfactants and may include a period of heating to aid in priondecontamination. In one embodiment, the concentration of surfactant usedwhen decontaminating prions may range from 0.01 to 50 wt %, preferably0.1 to 10 wt %. The heat treatment step typically occurs at atemperature of at least 40° C. to 130° C., preferably at least 80° C.,and most preferably at least 100° C. The optional heat treatmenttypically comprises a time of at least 1 minute up to 48 hours,preferably less than 24 hours, and most preferably less than 2 hours. Inone embodiment, the method to degrade prions includes both a surfactantand a heat treatment. Additionally, the proteases may be used incombination with an added lipase at a pH where the protease(s) alone maynot produce an efficacious concentration of peracid (e.g., at pH 4.0),the use of a synergistic combination of protease and lipase can producea concentration of peracid greater than the sum of peracid produced byeither protease or lipase alone.

The process to decontaminate a locus contaminated with (or at leastsuspected of being contaminated with) an infectious prion using thepresent aqueous peracid solutions may additionally include at least onestep of treating the locus with a surfactant before or after contactingthe locus with the aqueous peracid solution produced by the presentprocess. In another embodiment, the surfactant may optionally beincluded in the peracid reaction mixture prior to contacting the prioncontaminated locus. In yet another embodiment, the present process mayoptionally include a heat treatment step where the locus is heat treatedbefore, during, or after contacting said locus with the aqueous peracidsolution. In still yet another embodiment, the present process mayinclude both the optional heating step and the surfactant treatmentstep.

General Methods

The following examples are provided to demonstrate preferred aspects ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

All reagents and materials were obtained from DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), TCI America (Portland,Oreg.), Charkit Chemical Corporation (Darien, Conn.), Eastman ChemicalCo. (Kingsport, Tenn.) or Sigma/Aldrich Chemical Company (St. Louis,Mo.) unless otherwise specified. Enzymes were obtained fromSigma/Aldrich Chemical Company (St. Louis, Mo.), BioCatalytics(Pasadena, Calif.), Amano Enzymes USA (Lombard, Ill.), Valley Research(South Bend, Ind.), Enzyme Development Corporation (New York, N.Y.), andNovozymes (Franklinton, N.C.).

The abbreviations in the specification correspond to units of measure,techniques, properties, or compounds as follows: “sec” means second(s),“min” means minute(s), “h” or “hr” means hour(s), “d” means density ing/mL, “μL” means microliters, “mL” means milliliters, “L” means liters,“mM” means millimolar, “M” means molar, “mmol” means millimole(s), “wt”means weight, “wt %” means weight percent, “g” means grams, “μg” meansmicrograms, HPLC” means high performance liquid chromatography, “O.D.”means optical density at the designated wavelength, “dcw” means dry cellweight, “CFU” means colony forming units, “ATCC” means American TypeCulture Collection, “U” means units of perhydrolase activity, “RPM”means revolutions per minute, “EDTA” means ethylenediaminetetraaceticacid, “dd” means distilled and deionized, and “DTT” meansdithiothreitol.

Example 1 Lipase-Catalyzed Perhydrolysis of Triacetin at pH 6.5

Into a 4-mL glass vial with stir bar was added 1 mg enzyme (ICR 101-117,BioCatalytics, Pasadena, Calif.) in 0.050 mL of 50 mM potassiumphosphate buffer (pH 6.5), 0.900 mL of 278 mM triacetin in 50 mMpotassium phosphate buffer (pH 6.5, 250 mM final triacetinconcentration), and 0.052 mL 30% hydrogen peroxide (500 mM finalconcentration). After stirring for 5 or 30 minutes at 22° C., a 0.250 mLsample was filtered using a 30,000 Nominal Molecular Weight Limit (NMWL)filter (Millipore UltraFree-MC, Millipore Corp., Billerica, Mass.)centrifuged for 2 minute at 12,000 RPM. A portion of the filteredreaction samples was diluted 1:10 with dd water and analyzed forhydrogen peroxide, and the remaining portion of the sample was directlyanalyzed for peracid using the HPLC assay method (Table 1).

TABLE 1 Lipase-catalyzed Perhydrolysis of 250 mM Triacetin at pH 6.5.peracetic acid peracetic acid enzyme enzyme source (ppm), 5 min (ppm),30 min ICR-101 Aspergillus sp. 282 179 ICR-102 Rhizopus sp. 80 235ICR-103 Rhizopus oryzae 113 36 ICR-104 Penicillium sp. I 131 142 ICR-105Penicillium sp. II 144 66 ICR-106 Candida rugosa 171 105 ICR-107Pseudomonas cepacia 179 0 ICR-108 Pseudomonas sp. 180 30 ICR-109Pseudomonas fluorescens 183 11 ICR-110 Candida antartica lipase B 337398 ICR-111 Candida sp. 238 117 ICR-112 Candida antartica lipase A 134 0ICR-113 Pseudomonas sp. 241 358 ICR-114 porcine pancreas 135 0 ICR-115T. languinosus 216 113 ICR-116 Mucor miehei 238 117 ICR-117 Alcaligenessp. 293 370

Example 2 Lipase-Catalyzed Perhydrolysis of Triacetin at pH 4.0

Into a 4-mL glass vial with stir bar was added 1 mg enzyme (ICR 101-117,BioCatalytics, Pasadena, Calif.) in 0.050 mL of 50 mM sodiumacetate/acetic acid buffer (pH 4.0), 0.900 mL of 278 mM triacetin in 50mM sodium acetate/acetic acid buffer (pH 4.0, 250 mM final triacetinconcentration), and 0.052 mL 30% hydrogen peroxide (500 mM finalconcentration). After stirring for 5 or 30 minutes at 22° C., a 0.250 mLsample was filtered using a 30,000 NMWL filter (Millipore UltraFree-MC)centrifuged for 2 minute at 12,000 RPM. A portion of the filteredreaction samples was diluted 1:10 with dd water and analyzed forhydrogen peroxide, and the remaining portion of the sample was directlyanalyzed for peracid using the HPLC assay method (Table 2).

TABLE 2 Lipase-catalyzed Perhydrolysis of 250 mM Triacetin at pH 4.0.peracetic acid peracetic acid enzyme enzyme source (ppm), 5 min (ppm),30 min no enzyme 10 9.6 ICR-101 Aspergillus sp. 238 555 ICR-102 Rhizopussp. 11 0 ICR-103 Rhizopus oryzae 0 4 ICR-104 Penicillium sp. I 9 16ICR-105 Penicillium sp. II 11 19 ICR-106 Candida rugosa 23 34 ICR-107Pseudomonas cepacia 32 135 ICR-108 Pseudomonas sp. 33 53 ICR-109Pseudomonas fluorescens 32 68 ICR-110 Candida antartica lipase B 362 688ICR-111 Candida sp. 24 79 ICR-112 Candida antartica lipase A 151 86ICR-113 Pseudomonas sp. 221 370 ICR-114 porcine pancreas 165 53 ICR-115T. languinosus 206 132 ICR-116 Mucor miehei 14 0 ICR-117 Alcaligenes sp.185 710

Example 3 Lipase-Catalyzed Perhydrolysis of Triacetin at pH 4.0

Into a 4-mL glass vial with stir bar was added 2 mg enzyme (lipase ICR101 or ICR 110, BioCatalytics, Pasadena, Calif.) and 1.0 mL of 50 mMsodium acetate/acetic acid buffer (pH 4.0) containing from 250 mM or 500mM triacetin and 500 mM or 2500 mM hydrogen peroxide). After stirringfor 5 or 30 minutes at 23° C., a 0.250 mL sample was filtered using a30,000 NMWL filter (Millipore UltraFree-MC) centrifuged for 2 minute at12,000 RPM. A portion of the filtered reaction sample was diluted 1:20with dd water and analyzed for peracid using the HPLC assay method(Table 3).

TABLE 3 Lipase-catalyzed Perhydrolysis of 250 mM or 500 mM Triacetin atpH 4.0. triacetin H₂O₂ peracetic acid peracetic acid enzyme (mM) (mM)(ppm), 5 min (ppm), 30 min no enzyme 250 500 10 10 ICR-101 250 500 4611030 ICR-110 250 500 498 568 no enzyme 250 2500 17 15 ICR-101 250 2500656 1158 ICR-110 250 2500 1570 2880 no enzyme 500 2500 10 207 ICR-101500 2500 612 1109 ICR-110 500 2500 1860 3755

Example 4 Lipase-Catalyzed Perhydrolysis of a Mixture of Diacetin,Triacetin and Monoacetin at pH 6.5

Into a 4-mL glass vial with stir bar was added 1 mg enzyme (ICR 101-117,BioCatalytics, Pasadena, Calif.) in 0.050 mL of 50 mM potassiumphosphate buffer (pH 6.5), 0.900 mL of an aqueous mixture containingdiacetin (278 mM), triacetin (144 mM) and monoacetin (131 mM) in 50 mMpotassium phosphate buffer (pH 6.5), and either 0.052 mL 30% hydrogenperoxide (500 mM final concentration) or 0.026 mL 30% hydrogen peroxide(250 mM final concentration). After stirring for 5 or 30 minutes at 22°C., a 0.250 mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) centrifuged for 2 minute at 12,000 RPM. A portion of thefiltered reaction samples was diluted 1:10 with dd water and analyzedfor hydrogen peroxide, and the remaining portion of the sample wasdirectly analyzed for peracid using the HPLC assay method (Table 4).

TABLE 4 Lipase-catalyzed Perhydrolysis of a Mixture of Diacetin (278mM), Triacetin (144 mM) and Monoacetin (131 mM) at pH 6.5. 500 mM H₂O₂peracetic 250 mM H₂O₂ 250 mM H₂O₂ 500 mM H₂O₂ acid peracetic acidperacetic acid peracetic acid (ppm), enzyme (ppm), 5 min (ppm), 30 min(ppm), 5 min 30 min no enzyme 16 56 19 44 ICR-101 136 250 194 277ICR-102 127 159 26 232 ICR-103 139 133 275 223 ICR-104 151 174 284 286ICR-105 0 0 0 0 ICR-106 0 16 29 47 ICR-107 0 102 17 54 ICR-108 3 27 0 7ICR-109 0 38 38 0 ICR-110 451 384 669 484 ICR-111 0 21 12 43 ICR-112 024 20 38 ICR-113 34 202 80 236 ICR-114 0 0 0 0 ICR-115 0 8 11 8 ICR-1160 7 0 0 ICR-117 114 353 61 456

Example 5 Lipase-Catalyzed Perhydrolysis of a Mixture of Diacetin,Triacetin and Monoacetin at pH 4.0

Into a 4-mL glass vial with stir bar was added 1 mg enzyme (ICR 101,110, 113, or 117, BioCatalytics, Pasadena, Calif.) in 0.050 mL of 50 mMsodium acetate/acetic acid buffer (pH 4.0), 0.900 mL of an aqueousmixture containing either a) diacetin (278 mM), triacetin (144 mM) andmonoacetin (131 mM) in 50 mM sodium acetate/acetic acid buffer (pH 4.0)or b) diacetin (131 mM), triacetin (68 mM) and monoacetin (62 mM) in 50mM sodium acetate/acetic acid buffer (pH 4.0), and 0.052 mL 30% hydrogenperoxide (500 mM final concentration). After stirring for 5 or 30minutes at 22° C., a 0.250 mL sample was filtered using a 30,000 NMWLfilter (Millipore UltraFree-MC) centrifuged for 2 minute at 12,000 RPM.A portion of the filtered reaction samples was diluted 1:10 with ddwater and analyzed for hydrogen peroxide, and the remaining portion ofthe sample was directly analyzed for peracid using the HPLC assay method(Table 5).

TABLE 5 Lipase-catalyzed Perhydrolysis of Mixtures of Diacetin,Triacetin and Monoacetin at pH 4.0. substrate concentration diacetindiacetin (131 mM), (278 mM), triacetin triacetin (68 mM), (144 mM)monoacetin monoacetin (62 mM) (131 mM) peracetic peracetic acidperacetic acid acid peracetic acid enzyme (ppm), 5 min (ppm), 30 min(ppm), 5 min (ppm), 30 min no enzyme 4 2 1 2 ICR-101 183 457 ICR-110 502616 982 948 ICR-113 102 358 ICR-117 280 896

Example 6 Protease-Catalyzed Perhydrolysis of Acetamide and Diacetamideat pH 4.0

Into a 4-mL glass vial with stir bar was added 1 mg protease (Table 6)in 0.050 mL of 50 mM sodium acetate/acetic acid buffer (pH 4.0), 0.900mL of an aqueous mixture containing either a) acetamide (278 mM) in 50mM sodium acetate/acetic acid buffer (pH 4.0) or b) diacetamide (278 mM)in 50 mM sodium acetate/acetic acid buffer (pH 4.0), and 0.052 mL 30%hydrogen peroxide (500 mM final concentration). After stirring for 5 or30 minutes at 22° C., a 0.250 mL sample was filtered using a 30,000 NMWLfilter (Millipore UltraFree-MC) centrifuged for 2 minute at 12,000 RPM.A portion of the filtered reaction samples was diluted 1:10 with ddwater and analyzed for hydrogen peroxide, and the remaining portion ofthe sample was directly analyzed for peracid using the HPLC assay method(Table 7).

TABLE 6 Protease and Supplier. catalog enzyme, source number supplierProtease Type XIII, Aspergillus saitoi P2143 Sigma Protease Type XVIII,Rhizopus sp. P5027 Sigma Protease, Bacillus sp. P5985 Sigma Pepsin,porcine stomach P6887 Sigma Chymopapain, papaya latex C8526 SigmaBromelain, pineapple stem 3000 GDU Hong Mao Biochemicals Papain, papayalatex P3125 Sigma Pronase, S. griseus 81748 Biochemika Proteinase K, T.album, 3115879 Roche recombinantly expressed in P. pastoris ProteinaseK, T. album 70663 Novagen

TABLE 7 Protease-catalyzed Perhydrolysis of Acetamide (250 mM) andDiacetamide (250 mM) at pH 4.0. acetamide acetamide diacetamidediacetamide (250 mM) (250 mM) (250 mM) (250 mM) peracetic acid peraceticacid peracetic acid peracetic acid protease (ppm), 5 min (ppm), 30 min(ppm), 5 min (ppm), 30 min no enzyme (control) 0 0 4 16 Protease TypeXIII 35 49 88 116 Protease Type XVIII 27 41 94 107 Protease, Bacillussp. 48 61 95 138 Pepsin 45 65 97 120 Chymopapain 66 65 100 131 Bromelain57 63 92 130 Papain 81 105 135 172 Pronase 2 43 Proteinase K, T. album,26 43 recombinantly expressed in P. pastoris Proteinase K, T. album 3449

Example 7 Protease-Catalyzed Perhydrolysis of Triacetin at pH 4.0

Into a 4-mL glass vial with stir bar was added 1 mg protease (Table 6)in 0.050 mL of 50 mM sodium acetate/acetic acid buffer (pH 4.0), 0.900mL of an aqueous mixture containing triacetin (278 mM) in 50 mM sodiumacetate/acetic acid buffer (pH 4.0), and 0.052 mL 30% hydrogen peroxide(500 mM final concentration). After stirring for 5 or 30 minutes at 22°C., a 0.250 mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) centrifuged for 2 minute at 12,000 RPM. A portion of thefiltered reaction samples was diluted 1:10 with dd water and analyzedfor hydrogen peroxide, and the remaining portion of the sample wasdirectly analyzed for peracid using the HPLC assay method (Table 8).

TABLE 8 Protease-catalyzed Perhydrolysis of Triacetin (250 mM) at pH4.0. triacetin triacetin (250 mM) (250 mM) peracetic acid peracetic acidprotease (ppm), 5 min (ppm), 30 min no enzyme (control) 10 10 ProteaseType XIII 209 358 Protease Type XVIII 65 30 Protease, Bacillus sp. 74 55Pepsin 65 44 Chymopapain 72 57 Bromelain 83 59 Papain 85 48

Example 8 Protease-Catalyzed Perhydrolysis of Triacetin at pH 6.5

Into a 4-mL glass vial with stir bar was added 1 mg protease (Table 6)in 0.050 mL of 50 mM sodium phosphate buffer (pH 6.5), 0.900 mL of anaqueous mixture containing triacetin (278 mM) in 50 mM phosphate buffer(pH 6.5), and 0.052 mL 30% hydrogen peroxide (500 mM finalconcentration). After stirring for 5 or 30 minutes at 22° C., a 0.250 mLsample was filtered using a 30,000 NMWL filter (Millipore UltraFree-MC)centrifuged for 2 minute at 12,000 RPM. A portion of the filteredreaction samples was diluted 1:10 with dd water and analyzed forhydrogen peroxide, and the remaining portion of the sample was directlyanalyzed for peracid using the HPLC assay method (Table 9).

TABLE 9 Protease-catalyzed Perhydrolysis of Triacetin (250 mM) at pH6.5. triacetin triacetin (250 mM) (250 mM) peracetic acid peracetic acidprotease (ppm), 5 min (ppm), 30 min no enzyme (control) 51 26 ProteaseType XIII 135 136 Protease Type XVIII 89 119 Protease, Bacillus sp. 9364 Pepsin 111 163 Chymopapain 69 167 Bromelain 161 170 Papain 203 142

Example 9 Protease-Catalyzed Perhydrolysis of Acetamide and Diacetamideat pH 6.5

Into a 4-mL glass vial with stir bar was added 1 mg protease (Table 6)in 0.050 mL of 50 mM potassium phosphate buffer (pH 6.5), 0.900 mL of anaqueous mixture containing either a) acetamide (278 mM) in 50 mMpotassium phosphate buffer (pH 6.5) or b) diacetamide (278 mM) in 50 mMpotassium phosphate buffer (pH 6.5), and 0.052 mL 30% hydrogen peroxide(500 mM final concentration). After stirring for 5 or 30 minutes at 22°C., a 0.250 mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) centrifuged for 2 minute at 12,000 RPM. A portion of thefiltered reaction samples was diluted 1:10 with dd water and analyzedfor hydrogen peroxide, and the remaining portion of the sample wasdirectly analyzed for peracid using the HPLC assay method (Table 10).

TABLE 10 Protease-catalyzed Perhydrolysis of Acetamide (250 mM) andDiacetamide (250 mM) at pH 6.5. acetamide acetamide diacetamidediacetamide (250 mM) (250 mM) (250 mM) (250 mM) peracetic acid peraceticacid peracetic acid peracetic acid protease (ppm), 5 min (ppm), 30 min(ppm), 5 min (ppm), 30 min no enzyme (control) 23 26 59 62 Pronase 126168 17 17 Proteinase K, T. album, 150 194 66 99 recombinantly expressedin P. pastoris Proteinase K, T. album 270 214 104 100 no enzyme(control) 21 0 0 33 Protease Type XIII 42 32 218 206 Protease Type XVIII58 33 226 180 Protease, Bacillus sp. 89 73 807 181 Pepsin 81 67 259 198Chymopapain 94 12 218 170 Bromelain 108 112 311 202 Papain 199 157 342187

Example 10 Enzymatic Perhydrolysis of Triacetin Using a Combination ofLipase and Proteases at pH 4.0 and 6.5

Reaction mixtures were prepared containing 1 mg lipase (ICR 101,ICR-110, or ICR-117, BioCatalytics, Pasadena, Calif.), 0.3 mg ProteinaseK (T. album), and 1.2 mg of Pronase in 1.0 mL of 50 mM buffer (eithersodium acetate/acetic acid buffer at pH 4.0, or potassium phosphatebuffer at pH 6.5), additionally containing triacetin (250 mM) andhydrogen peroxide (500 mM). After stirring for 5 or 30 minutes at 22°C., a 0.250 mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) centrifuged for 2 minute at 12,000 RPM. A portion of thefiltered reaction samples was diluted 1:10 with dd water and analyzedfor hydrogen peroxide, and the remaining portion of the sample wasdirectly analyzed for peracid using the HPLC assay method (Table 11).Reactions were also run with lipase alone or protease(s) alone forcomparison of peracetic acid concentrations produced without thecombination of lipase and protease(s).

TABLE 11 Enzymatic Perhydrolysis of Triacetin (250 mM) using aCombination of Lipase and Proteases at pH 4.0 and 6.5. peracetic acidperacetic acid enzyme source pH (ppm), 5 min (ppm), 30 min no enzyme(control) 4.0 10 10 Proteinase K 4.0 24 7 Pronase 4.0 17 5 ProteinaseK/Pronase 4.0 8 15 ICR-101 4.0 238 555 Proteinase K/Pronase/ICR101 4.0252 542 ICR-110 4.0 362 688 Proteinase K/Pronase/ICR110 4.0 460 679ICR-117 4.0 284 927 Proteinase K/Pronase/ICR117 4.0 401 909 no enzyme(control) 6.5 22 59 Proteinase K 6.5 323 669 Pronase 6.5 114 484Proteinase K/Pronase 6.5 225 433 ICR-101 6.5 306 588 ProteinaseK/Pronase/ICR101 6.5 359 576 ICR-110 6.5 337 398 ProteinaseK/Pronase/ICR110 6.5 406 364 ICR-117 6.5 438 984 ProteinaseK/Pronase/ICR117 6.5 579 1189

Example 11 (Comparative) Enzymatic Perhydrolysis of Propyl Acetate orTriacetin at pH 6.5

Into a 4-mL glass vial with stir bar was added 2 mg of one of thefollowing enzymes: acetylcholinesterase (Sigma, C-2888), Lipase G“Amano” 50 (Amano), or Chirazyme L2, lyo. (C. antartica lipase B,BioCatalytics) dissolved in 0.050 mL of 50 mM potassium phosphate buffer(pH 6.5). To the vial was then added either a) 0.930 mL of a solutioncontaining 50 mM potassium phosphate buffer (pH 6.5) and 215 mM propylacetate, followed by 0.021 mL 30% hydrogen peroxide (200 mM finalhydrogen peroxide concentration) or b) 0.900 mL of a solution containing278 mM triacetin in 50 mM potassium phosphate buffer (pH 6.5), 0.024 mLof 50 mM potassium phosphate buffer (pH 6.5), and 0.026 mL 30% hydrogenperoxide (250 mM final concentration). After stirring for 5 or 30minutes at 22° C., a 0.250 mL sample of the reaction mixture wasfiltered using a 30,000 NMWL filter (Millipore UltraFree-MC) centrifugedfor 2 minute at 12,000 RPM. A portion of the resulting filtrate wasdiluted 1:10 with dd water and analyzed for hydrogen peroxide, and theremaining portion of the filtrate was directly analyzed for peracidusing the HPLC assay method (Table 12).

TABLE 12 Enzymatic Perhydrolysis of Propyl Acetate (200 mM) or Triacetin(250 mM) at pH 6.5. propyl acetate propyl acetate triacetin triacetin(200 mM), (200 mM), (250 mM), (250 mM) H₂O₂ H₂O₂ H₂O₂ H₂O₂ (200 mM) (200mM) (250 mM) (250 mM) peracetic acid peracetic acid peracetic acidperacetic acid enzyme (ppm), 5 min (ppm), 30 min (ppm), 5 min (ppm), 30min no enzyme (control) 12 69 22 70 acetyl cholinesterase 0 0 Lipase G“Amano” 50 18 46 78 Chirazyme L2 19 1 192 173

Example 12 Lipase-Catalyzed Perhydrolysis of a Mixture of Diacetin,Triacetin and Monoacetin at pH 6.5

A 1-mL reaction mixture was prepared containing 2 mg, 1 mg or 0.2 mg ofenzyme (A “Amano” 12 lipase (A. niger lipase, Amano), Validase lipase AN(A. niger lipase, Valley Research), ICR 110 (C. antartica lipase,BioCatalytics), Dietrenz CR (C. rugosa lipase, Valley Research), AmanoF-DS (R. oryzae lipase, Amano), Amano DS (A. niger lipase, Amano), orEnzeco MLC (Microbial Lipase Concentrate, A. niger lipase, EnzymeDevelopment Corporation)) dissolved in 1.0 mL of 50 mM potassiumphosphate buffer (pH 6.5) containing a mixture of diacetin (236 mM),triacetin (83 mM) and monoacetin (181 mM) and either 2500 mM or 1000 mMhydrogen peroxide. After stirring for 5 or 30 minutes at 25° C., a 0.250mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) for 2 minute at 12,000 RPM. A portion of the filteredreaction sample was diluted 1:25 with dd water and analyzed for peracidusing the HPLC assay method (Table 13).

TABLE 13 Lipase-catalyzed Perhydrolysis of Diacetin (236 mM), Triacetin(83 mM) and Monoacetin (181 mM) at pH 6.5. [enzyme] H₂O₂ peracetic acidperacetic acid enzyme (mg/mL) (mM) (ppm), 5 min (ppm), 30 min no enzyme0 2500 80 60 A“Amano”12 2 2500 460 520 Validase AN 2 2500 590 510ICR-110 2 2500 2290 2350 A“Amano”12 1 2500 420 350 Validase AN 1 2500190 390 ICR-110 1 2500 2100 2240 ICR-110 0.2 2500 610 1850 no enzyme 01000 90 110 A“Amano”12 2 1000 310 580 Validase AN 2 1000 340 550 ICR-1102 1000 1240 1030 A“Amano”12 1 1000 240 525 Validase AN 1 1000 220 470ICR-110 1 1000 335 595 Dietrenz CR 1 1000 70 240 Amano F-DS 1 1000 10 10Amano DS 1 1000 170 430 Enzeco MLC 1 1000 155 390 ICR-110 0.2 1000 380410 no enzyme 0 500 40 30 A“Amano”12 1 500 215 490 Validase AN 1 500 120500 ICR-110 1 500 240 490 Dietrenz CR 1 500 0 210 Amano F-DS 1 500 35 75Amano DS 1 500 60 305 Enzeco MLC 1 500 145 405

Example 13 Lipase-Catalyzed Perhydrolysis of a Mixture of Diacetin,Triacetin and Monoacetin at pH 4.0

A 1-mL reaction mixture was prepared containing 2 mg, 1 mg or 0.2 mg ofenzyme (A “Amano” 12 lipase (A. niger lipase, Amano), Validase lipase AN(A. niger lipase, Valley Research), or ICR 110 (C. antartica lipase,BioCatalytics)) dissolved in 1.0 mL of 50 mM sodium acetate/acetic acidbuffer (pH 4.0) containing a mixture of diacetin (236 mM), triacetin (83mM) and monoacetin (181 mM) and either 2500 mM or 1000 mM hydrogenperoxide. After stirring for 5 or 30 minutes at 25° C., a 0.250 mLsample was filtered using a 30,000 NMWL filter (Millipore UltraFree-MC)for 2 minute at 12,000 RPM. A portion of the filtered reaction samplewas diluted 1:25 with dd water and analyzed for peracid using the HPLCassay method (Table 14).

TABLE 14 Lipase-catalyzed Perhydrolysis of Diacetin (236 mM), Triacetin(83 mM) and Monoacetin (181 mM) at pH 4.0. [enzyme] H₂O₂ peracetic acidperacetic acid enzyme (mg/mL) (mM) (ppm), 5 min (ppm), 30 min no enzyme0 2500 10 40 A“Amano”12 2 2500 870 605 Validase AN 2 2500 510 560ICR-110 2 2500 1670 2250 A“Amano”12 1 2500 240 700 Validase AN 1 2500270 600 ICR-110 1 2500 1740 2180 ICR-110 0.2 2500 480 1470 no enzyme 01000 10 10 A“Amano”12 2 1000 320 600 Validase AN 2 1000 270 690 ICR-1102 1000 1080 920 ICR-110 0.2 1000 870 1060

Example 14 Lipase-Catalyzed Perhydrolysis of a Mixture of Diacetin,Triacetin and Monoacetin at pH 6.5

A 1-mL reaction mixture was prepared containing 2 mg or 1 mg of enzyme(A “Amano” 12 lipase (A. niger lipase, Amano), Validase lipase AN (A.niger lipase, Valley Research), or ICR 110 (C. antartica lipase,BioCatalytics)) dissolved in 1.0 mL of 50 mM potassium phosphate buffer(pH 6.5) containing a mixture of diacetin (500 mM), triacetin (176 mM)and monoacetin (383 mM), and either 2500 mM or 1000 mM hydrogenperoxide. After stirring for 5 or 30 minutes at 25° C., a 0.250 mLsample was filtered using a 30,000 NMWL filter (Millipore UltraFree-MC)for 2 minute at 12,000 RPM. A portion of the filtered reaction samplewas diluted 1:25 with dd water and analyzed for peracid using the HPLCassay method (Table 15).

TABLE 15 Lipase-catalyzed Perhydrolysis of a Mixture of Diacetin (500mM), Triacetin (176 mM) and Monoacetin (383 mM) at pH 6.5. [enzyme] H₂O₂peracetic acid peracetic acid enzyme (mg/mL) (mM) (ppm), 5 min (ppm), 30min no enzyme 0 2500 11 13 A“Amano”12 2 2500 394 860 Validase AN 2 2500354 650 ICR-110 2 2500 5224 7130 A“Amano”12 1 2500 290 710 Validase AN 12500 220 450 ICR-110 1 2500 3290 6130 no enzyme 0 1000 70 50 A“Amano”122 1000 420 930 Validase AN 2 1000 490 890 ICR-110 2 1000 2900 2600

Example 15 Lipase-Catalyzed Perhydrolysis of a Mixture of Diacetin,Triacetin and Monoacetin at pH 4.0

A 1-mL reaction mixture was prepared containing 2 mg or 1 mg of enzyme(A “Amano” 12 lipase (A. niger lipase, Amano), Validase lipase AN (A.niger lipase, Valley Research), or ICR 110 (C. antartica lipase,BioCatalytics)) dissolved in 1.0 mL of 50 mM sodium acetate/acetic acidbuffer (pH 4.0) containing a mixture of diacetin (500 mM), triacetin(176 mM) and monoacetin (383 mM), and either 2500 mM or 1000 mM hydrogenperoxide. After stirring for 5 or 30 minutes at 25° C., a 0.250 mLsample was filtered using a 30,000 NMWL filter (Millipore UltraFree-MC)for 2 minute at 12,000 RPM. A portion of the filtered reaction samplewas diluted 1:25 with dd water and analyzed for peracid using the HPLCassay method (Table 16).

TABLE 16 Lipase-catalyzed Perhydrolysis of a mixture of Diacetin (500mM), Triacetin (176 mM) and Monoacetin (383 mM) at pH 4.0. [enzyme] H₂O₂peracetic acid peracetic acid enzyme (mg/mL) (mM) (ppm), 5 min (ppm), 30min no enzyme 0 2500 10 10 A“Amano”12 2 2500 380 920 Validase AN 2 2500440 930 ICR-110 2 2500 5760 7210 A“Amano”12 1 2500 180 480 Validase AN 12500 170 520 ICR-110 1 2500 3960 7450 no enzyme 0 1000 10 10 A“Amano”122 1000 380 900 Validase AN 2 1000 400 1150 ICR-110 2 1000 2970 2510

Example 16 Lipase-Catalyzed Perhydrolysis of Triacetin at pH 6.5

A 1-mL reaction mixture was prepared containing 2 mg, 1 mg or 0.2 mg ofenzyme (A “Amano” 12 lipase (A. niger lipase, Amano), Validase lipase AN(A. niger lipase, Valley Research), and ICR 110 (C. antartica lipase,BioCatalytics)) dissolved in 1.0 mL of 50 mM potassium phosphate buffer(pH 6.5) containing 500 mM triacetin and either 2500 mM or 1000 mMhydrogen peroxide. After stirring for 5 or 30 minutes at 25° C., a 0.250mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) for 2 minute at 12,000 RPM. A portion of the filteredreaction sample was diluted 1:25 with dd water and analyzed for peracidusing the HPLC assay method (Table 17).

TABLE 17 Lipase-catalyzed Perhydrolysis of Triacetin (500 mM) at pH 6.5.[enzyme] H₂O₂ peracetic acid peracetic acid enzyme (mg/mL) (mM) (ppm), 5min (ppm), 30 min no enzyme 0 2500 80 170 A“Amano”12 2 2500 800 1390Validase AN 2 2500 790 1180 ICR-110 2 2500 2710 2880 A“Amano”12 1 2500670 1070 Validase AN 1 2500 460 810 ICR-110 1 2500 1840 2421 ICR-110 0.22500 1120 760 no enzyme 0 1000 130 80 A“Amano”12 2 1000 970 1250Validase AN 2 1000 770 1310 ICR-110 2 1000 760 1400 ICR-110 0.2 1000 450680

Example 17 Lipase-Catalyzed Perhydrolysis of Triacetin at pH 4.0

A 1-mL reaction mixture was prepared containing 2 mg, 1 mg or 0.2 mg ofenzyme (A “Amano” 12 lipase (A. niger lipase, Amano), Validase lipase AN(A. niger lipase, Valley Research), or ICR 110 (C. antartica lipase,BioCatalytics)) dissolved in 1.0 mL of 50 mM sodium acetate/acetic acidbuffer (pH 4.0) containing triacetin (500 mM) and either 2500 mM or 1000mM hydrogen peroxide. After stirring for 5 or 30 minutes at 25° C., a0.250 mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) for 2 minute at 12,000 RPM. A portion of the filteredreaction sample was diluted 1:25 with dd water and analyzed for peracidusing the HPLC assay method (Table 18).

TABLE 18 Lipase-catalyzed Perhydrolysis of Triacetin (500 mM) at pH 4.0.[enzyme] H₂O₂ peracetic acid peracetic acid enzyme (mg/mL) (mM) (ppm), 5min (ppm), 30 min no enzyme 0 2500 10 20 A“Amano”12 2 2500 1030 1370Validase AN 2 2500 920 840 ICR-110 2 2500 2810 3700 A“Amano”12 1 2500570 740 Validase AN 1 2500 570 680 ICR-110 1 2500 2120 2860 ICR-110 0.22500 510 780 no enzyme 0 1000 5 5 A“Amano”12 2 1000 1100 1520 ValidaseAN 2 1000 930 1230 ICR-110 2 1000 1540 1670 ICR-110 0.2 1000 370 600

Example 18 CALB L-Catalyzed Perhydrolysis of Triacetin at pH 4.0 and pH6.5

A 1-mL reaction mixture was prepared containing 0.010 mL of CALB Llipase (a commercial liquid formulation of C. antartica lipase B,Novozymes) dissolved in either 1.0 mL of 50 mM sodium acetate/aceticacid buffer (pH 4.0) or 1.0 mL of 50 mM potassium phosphate buffer (pH6.5) additionally containing triacetin (250 mM) and hydrogen peroxide(2500 mM). After stirring for 5 or 30 minutes at 25° C., a 0.250 mLsample was filtered using a 30,000 NMWL filter (Millipore UltraFree-MC)for 2 minute at 12,000 RPM. A portion of the filtered reaction samplewas diluted 1:20 with dd water and analyzed for peracid using the HPLCassay method (Table 19).

TABLE 19 Lipase-catalyzed Perhydrolysis of Triacetin (250 mM) at pH 4.0or pH 6.5 using CALB L. H₂O₂ peracetic acid peracetic acid enzyme pH(mM) (ppm), 5 min (ppm), 30 min no enzyme 4.0 2500 28 15 CALB L 4.0 2500439 1120 no enzyme 6.5 2500 226 108 CALB L 6.5 2500 1294 678

Example 19 Perhydrolysis of Triacetin at pH 4.0 Using Immobilized C.antartica Lipase B

A reaction mixture was prepared containing 100 mg of IMB-111(immobilized Candida antartica lipase B, BioCatalytics) suspended in10.0 mL of 50 mM sodium acetate/acetic acid buffer (pH 4.0) additionallycontaining triacetin (500 mM) and hydrogen peroxide (2500 mM). Aftermixing on a rotating platform for a predetermined time from 2 to 30minutes at 25° C., a 0.100 mL sample of the liquid portion was filteredusing a 30,000 NMWL filter (Millipore UltraFree-MC) for 2 minute at12,000 RPM. A portion of the filtered reaction sample was diluted 1:20with dd water and analyzed for peracid using the HPLC assay method(Table 20).

TABLE 20 Lipase-catalyzed Perhydrolysis of Triacetin (500 mM) at pH 4.0using immobilized Candida Antartica lipase B (IMB-111). no enzyme, 10mg/mL IMB-111, Time peracetic acid peracetic acid (min) (ppm) (ppm) 2 42911 5 50 1667 10 49 2244 15 24 2755 20 43 3261 25 49 3369 30 57 3757

The above reaction was repeated with fresh catalyst, and after 30 min,the concentration of peracetic acid was 3621 ppm. The catalyst wasrecovered from the reaction mixture, washed twice with 10.0 mL of 50 mMsodium acetate/acetic acid buffer (pH 4.0), and the reaction wasrepeated using the recovered enzyme; after 30 min, the concentration ofperacetic acid was 2274 ppm.

Example 20 Timecourse for Enzymatic Perhydrolysis of Triacetin at pH 6.5

A reaction mixture containing 1.0 mg/mL of DS lipase (A. niger, Amano)dissolved in 50 mM potassium phosphate buffer (pH 6.5) containinghydrogen peroxide (2500 mM) and triacetin (500 mM) was stirred at 25° C.A 0.100 mL aliquot was withdrawn at pre-determined times and filteredusing a 30,000 NMWL filter (Millipore UltraFree-MC) for 2 minute at12,000 RPM. A portion of the filtered reaction sample was diluted 1:20with dd water and analyzed for peracid using the HPLC assay method(Table 21).

TABLE 21 Timecourse for DS Lipase-catalyzed Perhydrolysis of Triacetin(500 mM) at pH 6.5. no enzyme, 1 mg/mL DS lipase, Time peracetic acidperacetic acid (min or h) (ppm) (ppm) 5 min 10 367 30 min 149 522 2 h 26819 5 h 78 1481 19 h 27 1895

Example 21 Biocidal Activity of a Peracetic Acid Formulation Preparedfrom Triacetin, Hydrogen Peroxide and C. antartica Lipase B

A reaction was performed using Chirazyme L2 lipase (C. antartica lipaseB) as enzyme catalyst for the production of peracetic acid. Into a 20-mLglass vial equipped with magnetic stir bar was placed 9.00 mL of 278 mMtriacetin in 50 mM phosphate buffer (pH 6.5), 0.258 mL of 30% hydrogenperoxide, and 1.0 mL of 20 mg/mL Chirazyme L2 in 50 mM phosphate buffer(pH 6.5), and the resulting mixture stirred for 5 min at roomtemperature. The reaction was stopped by filtering the product mixtureusing a 30K NMWL filter unit (Millipore UltraFree-MC) centrifuged for 2minute at 12,000 rpm to remove the enzyme, and storing the filtrate onwet ice. The filtrate was analyzed for peracetic acid (PAA) and hydrogenperoxide using the HPLC assay method; control reactions were performedin the absence of added enzyme with and without triacetin. The filteredenzyme-generated product mixture contained 192 ppm PAA, produced fromboth the chemical and enzymatic reaction of triacetin with hydrogenperoxide; the hydrogen peroxide concentration in the product mixture was6431 ppm. The control reaction performed with 250-mM triacetin and noadded enzyme produced 22 ppm PAA generated from the chemical reaction oftriacetin and hydrogen peroxide.

The filtered enzyme-generated product mixture was diluted 1:5 withsterile water, and the resulting solution was further diluted withsterile water in four 1:1 serial dilutions to obtain the concentrationsof PAA and hydrogen peroxide listed in Table 22.

TABLE 22 Dilution Series Used to Assessing Biocidal Activity. dilutionfactor PAA (ppm) H₂O₂ (ppm) 1:5  38.3 1286 1:10 19.2 643 1:20 9.6 3221:40 4.8 161 1:80 2.4 80

The 1:5 dilution and four serial dilutions were evaluated for biocidalactivity by mixing 0.100 mL of the PAA-containing solution with 0.100 mLof an inoculum containing 2.7×10⁶ CFU/mL of E. coli ATCC 11229 (AmericanType Culture Collection, Manassas, Va.) in Millers LB broth diluted withButterfield Buffer; eight replicates of each mixture were contained inindividual wells of a sterile 96-well microtiter plate. After theresulting cell suspensions were allowed to stand for 10 minutes at roomtemperature, they were tested for viability using the growth indicatorXTT(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium,inner salt, monosodium salt; CAS RN 111072-31-2) according to the methodof Gabrielson et al. (J. Microbiol. Methods, 50:63-73 (2002)). No cellgrowth of 1.35×10⁶ CFU/mL (indicated by no OD measurement at 450 nmusing XTT) was observed with a Minimum Biocidal Concentration of PAA of2.4 ppm (six-log kill) (Table 23).

TABLE 23 Determination of Minimum Biocidal Concentration (MBC) ofPeracetic Acid Generated by Lipase-catalyzed Perhydrolysis of Triacetinfor E. coli ATCC 11229. control blank PAA (ppm) 19.15 9.6 4.8 2.4 1.2 0H₂O₂ (ppm) 643.1 321.6 160.8 80.4 40.2 0 replicate 1 OD (450 nm) 0.4030.427 0.418 0.406 0.692 2.177 0.418 replicate 2 OD (450 nm) 0.383 0.3980.393 0.400 0.674 2.142 0.415 replicate 3 OD (450 nm) 0.379 0.392 0.3880.397 0.635 2.007 0.414 replicate 4 OD (450 nm) 0.387 0.395 0.399 0.4030.658 2.114 0.418 replicate 5 OD (450 nm) 0.381 0.404 0.398 0.401 0.6252.171 0.409 replicate 6 OD (450 nm) 0.382 0.409 0.404 0.409 0.639 2.3470.405 replicate 7 OD (450 nm) 0.382 0.394 0.401 0.407 0.520 2.323 0.407replicate 8 OD (450 nm) 0.403 0.422 0.471 0.543 0.496 2.582 0.428 meanreplicate OD (450 nm) 0.388 0.405 0.409 0.421 0.617 2.233 0.414blank-corrected mean −0.029 −0.009 −0.005 0.007 0.203 1.819 0 OD:

Separately, the cell inoculum was treated with sterile solutions oftriacetin (Table 24) or hydrogen peroxide in 50 mM phosphate buffer (pH6.5) (Table 25) at the concentrations present in each of the biocidedilutions in Table 23 to determine the MBC for these components of thebiocide solution.

TABLE 24 Determination of Minimum Biocidal Concentration (MBC) ofTriacetin for E. coli ATCC 11229. control blank triacetin (ppm) 54552728 1364 682 341 0 replicate 1 OD (450 nm) 2.210 2.280 2.337 2.2132.408 2.375 0.401 replicate 2 OD (450 nm) 2.136 2.208 2.265 2.156 2.5072.397 0.420 replicate 3 OD (450 nm) 2.091 2.222 2.198 2.218 2.189 2.3860.402 replicate 4 OD (450 nm) 2.229 2.273 2.140 2.304 2.568 2.557 0.409replicate 5 OD (450 nm) 2.124 2.167 2.266 2.310 2.563 2.491 0.413replicate 6 OD (450 nm) 2.263 2.358 2.485 2.560 2.601 2.582 0.410replicate 7 OD (450 nm) 2.407 2.454 2.632 2.601 2.58 2.603 0.410replicate 8 OD (450 nm) 2.539 2.663 2.823 2.605 3.148 2.952 0.455 meanreplicate OD (450 nm) 2.250 2.328 2.393 2.371 2.571 2.543 0.415blank-corrected mean 1.835 1.913 1.978 1.956 2.156 2.128 0 OD:

TABLE 25 Determination of Minimum Biocidal Concentration (MBC) ofHydrogen Peroxide for E. coli ATCC 11229. control blank H₂0₂ (ppm) 680.5340.2 170.1 85.1 42.5 0 replicate 1 OD (450 nm) 0.456 0.440 0.401 1.8802.170 2.371 0.423 replicate 2 OD (450 nm) 0.436 0.428 0.422 1.793 2.0822.226 0.417 replicate 3 OD (450 nm) 0.446 0.424 0.470 1.834 2.034 2.1580.415 replicate 4 OD (450 nm) 0.433 0.423 0.435 1.889 2.007 2.216 0.422replicate 5 OD (450 nm) 0.440 0.439 0.402 1.815 2.027 2.372 0.417replicate 6 OD (450 nm) 0.426 0.425 0.424 1.795 1.971 2.581 0.419replicate 7 OD (450 nm) 0.430 0.432 0.462 1.962 2.117 2.741 0.442replicate 8 OD (450 nm) 0.453 0.452 0.414 2.153 2.666 2.821 0.439 meanreplicate OD (450 nm) 0.440 0.433 0.4295 1.890 2.134 2.436 0.424blank-corrected mean OD: 0.016 0.009 0.005 1.466 1.710 2.012 0

The MBC of PAA for E. coli ATCC 11229 was >2.4 ppm, measured in thepresence of 80 ppm of hydrogen peroxide (Table 23); the MBC for hydrogenperoxide was >170 ppm (Table 25), confirming that the biocidal activityof the PAA test solution was due to PAA and not H₂O₂. Triacetin had nobiocidal effect at the highest concentration examined (5455 ppm, Table24). The MBC for PAA generated enzymatically agrees closely with the MBCdetermined using a commercial source of peracetic acid (Table 26,MBC >2.3 ppm PAA).

TABLE 26 Determination of Minimum Biocidal Concentration (MBC) ofCommercially-Available Peracetic Acid for E. coli ATCC 11229. controlblank PAA (ppm) 18.75 9.4 4.7 2.3 1.15 0 replicate 1 OD (450 nm) 0.3060.301 0.310 0.303 1.734 2.098 0.305 replicate 2 OD (450 nm) 0.292 0.2960.297 0.288 1.367 1.832 0.295 replicate 3 OD (450 nm) 0.289 0.290 0.2940.292 1.343 1.816 0.295 replicate 4 OD (450 nm) 0.288 0.294 0.295 0.2911.377 1.874 0.292 replicate 5 OD (450 nm) 0.296 0.291 0.298 0.294 1.3681.869 0.294 replicate 6 OD (450 nm) 0.294 0.294 0.294 0.292 1.378 1.8740.290 replicate 7 OD (450 nm) 0.296 0.299 0.293 0.303 1.324 1.817 0.227replicate 8 OD (450 nm) 0.300 0.295 0.304 0.318 1.386 1.869 0.173 meanreplicate OD (450 nm) 0.295 0.295 0.298 0.298 1.410 1.881 0.271blank-corrected mean OD: 0.024 0.024 0.027 0.026 1.138 1.610 0

Example 22 Enzymatic Perhydrolysis of Triacetin Using a Combination ofLipase and Alcalase® at pH 4.0 and 6.5

Reaction mixtures were prepared containing 1 mg lipase (A “Amano” 12(Amano), Validase AN (Valley Research), or ICR-110 (BioCatalytics)), and20 μL Alcalase® (Novozymes) in 1.0 mL of 50 mM buffer (either sodiumacetate/acetic acid buffer at pH 4.0, or potassium phosphate buffer atpH 6.5), additionally containing triacetin (250 mM) and hydrogenperoxide (500 mM). After stirring for 5 or 30 minutes at 22° C., a0.250-mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) centrifuged for 2 minute at 12,000 RPM. A portion of thefiltered reaction samples was diluted 1:10 with dd water and analyzedfor hydrogen peroxide, and the remaining portion of the sample wasdirectly analyzed for peracid using the HPLC assay method (Table 27).Reactions were also run with lipase alone or Alcalase® alone forcomparison of peracetic acid concentrations produced without thecombination of lipase and Alcalase®.

TABLE 27 Enzymatic Perhydrolysis of Triacetin (250 mM) Using aCombination of Lipase and Alcalase ® at pH 4.0 and 6.5. peracetic acidperacetic acid enzyme source pH (ppm), 5 min (ppm), 30 min no enzyme(control) 4.0 10 10 Alcalase ® 4.0 0 0 A“Amano”12 4.0 300 610A“Amano”12/Alcalase ® 4.0 220 540 Validase AN 4.0 0 20 ValidaseAN/Alcalase ® 4.0 0 50 ICR-110 4.0 240 490 ICR110/Alcalase ® 4.0 330 580no enzyme (control) 6.5 80 90 Alcalase ® 6.5 180 330 A“Amano”12 6.5 460490 A“Amano”12/Alcalase ® 6.5 290 620 Validase AN 6.5 490 730 ValidaseAN/Alcalase ® 6.5 360 700 ICR-110 6.5 240 440 ICR110/Alcalase ® 6.5 370420

Example 23 Enzymatic Perhydrolysis of a Mixture of Diacetin (250 mM),Triacetin (88 mM) and Monoacetin (192 mM) Using a Combination of Lipaseand Alcalase® at pH 4.0 and 6.5

Reaction mixtures were prepared containing 1 mg lipase (A “Amano” 12(Amano), Validase AN (Valley Research), or ICR-110 (BioCatalytics)), and20 μL Alcalase® (Novozymes) in 1.0 mL of 50 mM buffer (either sodiumacetate/acetic acid buffer at pH 4.0, or potassium phosphate buffer atpH 6.5), additionally containing a mixture of diacetin (250 mM),triacetin (88 mM) and monoacetin (192 mM) and hydrogen peroxide (500mM). After stirring for 5 or 30 minutes at 22° C., a 0.250-mL sample wasfiltered using a 30,000 NMWL filter (Millipore UltraFree-MC) centrifugedfor 2 minute at 12,000 RPM. A portion of the filtered reaction sampleswas diluted 1:10 with dd water and analyzed for hydrogen peroxide, andthe remaining portion of the sample was directly analyzed for peracidusing the HPLC assay method (Table 28). Reactions were also run withlipase alone or Alcalase® alone for comparison of peracetic acidconcentrations produced without the combination of lipase and Alcalase®.

TABLE 28 Enzymatic Perhydrolysis of a mixture of Diacetin (250 mM),Triacetin (88 mM) and Monoacetin (192 mM) using a Combination of Lipaseand Alcalase ® at pH 4.0 and 6.5. peracetic acid peracetic acid enzymesource pH (ppm), 5 min (ppm), 30 min no enzyme (control) 4.0 19 44Alcalase ® 4.0 0 20 A“Amano”12 4.0 150 360 A“Amano”12/Alcalase ® 4.0 130330 Validase AN 4.0 0 80 Validase AN/Alcalase ® 4.0 0 10 ICR-110 4.0 670760 ICR110/Alcalase ® 4.0 710 780 no enzyme (control) 6.5 10 10Alcalase ® 6.5 110 210 A“Amano”12 6.5 190 580 A“Amano”12/Alcalase ® 6.5170 420 Validase AN 6.5 180 580 Validase AN/Alcalase ® 6.5 190 400ICR-110 6.5 360 500 ICR110/Alcalase ® 6.5 470 470

Example 24 Enzymatic Perhydrolysis of Triacetin Using a Combination ofLipase and Neutrase® at pH 4.0 and 6.5

Reaction mixtures were prepared containing 1 mg lipase (A “Amano” 12(Amano), Validase AN (Valley Research), or ICR-110 (BioCatalytics)), and20 μL Neutrase® (Novozymes) in 1.0 mL of 50 mM buffer (either sodiumacetate/acetic acid buffer at pH 4.0, or potassium phosphate buffer atpH 6.5), additionally containing triacetin (250 mM) and hydrogenperoxide (500 mM). After stirring for 5 or 30 minutes at 22° C., a0.250-mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) centrifuged for 2 minute at 12,000 RPM. A portion of thefiltered reaction samples was diluted 1:10 with dd water and analyzedfor hydrogen peroxide, and the remaining portion of the sample wasdirectly analyzed for peracid using the HPLC assay method (Table 29).Reactions were also run with lipase alone or Neutrase® alone forcomparison of peracetic acid concentrations produced without thecombination of lipase and Neutrase®.

TABLE 29 Enzymatic Perhydrolysis of Triacetin (250 mM) Using aCombination of Lipase and Neutrase ® at pH 4.0 and 6.5. peracetic acidperacetic acid enzyme source pH (ppm), 5 min (ppm), 30 min no enzyme(control) 4.0 10 10 Neutrase ® 4.0 0 0 A“Amano”12 4.0 300 610A“Amano”12/Neutrase ® 4.0 190 430 Validase AN 4.0 0 20 ValidaseAN/Neutrase ® 4.0 0 10 ICR-110 4.0 240 490 ICR110/Neutrase ® 4.0 190 430no enzyme (control) 6.5 125 110 Neutrase ® 6.5 110 300 A“Amano”12 6.5530 480 A“Amano”12/Neutrase ® 6.5 195 620 Validase AN 6.5 560 720Validase AN/Neutrase ® 6.5 330 600 ICR-110 6.5 310 430 ICR110/Neutrase ®6.5 300 390

Example 25 Enzymatic Perhydrolysis of a Mixture of Diacetin (250 mM),Triacetin (88 mM) and Monoacetin (192 mM) Using a Combination of Lipaseand Neutrase® at pH 4.0 and 6.5

Reaction mixtures were prepared containing 1 mg lipase (A “Amano” 12(Amano), Validase AN (Valley Research), or ICR-110 (BioCatalytics)), and20 μL Neutrase® (Novozymes) in 1.0 mL of 50 mM buffer (either sodiumacetate/acetic acid buffer at pH 4.0, or potassium phosphate buffer atpH 6.5), additionally containing a mixture of diacetin (250 mM),triacetin (88 mM) and monoacetin (192 mM) and hydrogen peroxide (500mM). After stirring for 5 or 30 minutes at 22° C., a 0.250-mL sample wasfiltered using a 30,000 NMWL filter (Millipore UltraFree-MC) centrifugedfor 2 minutes at 12,000 RPM. A portion of the filtered reaction sampleswas diluted 1:10 with dd water and analyzed for hydrogen peroxide, andthe remaining portion of the sample was directly analyzed for peracidusing the HPLC assay method (Table 30). Reactions were also run withlipase alone or Neutrase® alone for comparison of peracetic acidconcentrations produced without the combination of lipase and Neutrase®.

TABLE 30 Enzymatic Perhydrolysis of a mixture of Diacetin (250 mM),Triacetin (88 mM) and Monoacetin (192 mM) using a Combination of Lipaseand Neutrase ® at pH 4.0 and 6.5. peracetic acid peracetic acid enzymesource pH (ppm), 5 min (ppm), 30 min no enzyme (control) 4.0 35 10Neutrase ® 4.0 50 110 A“Amano”12 4.0 135 310 A“Amano”12/Neutrase ® 4.0145 320 Validase AN 4.0 0 30 Validase AN/Neutrase ® 4.0 125 400 ICR-1104.0 655 710 ICR110/Neutrase ® 4.0 515 500 no enzyme (control) 6.5 35 10Neutrase ® 6.5 50 110 A“Amano”12 6.5 245 460 A“Amano”12/Neutrase ® 6.5145 390 Validase AN 6.5 235 460 Validase AN/Neutrase ® 6.5 125 320ICR-110 6.5 415 380 ICR110/Neutrase ® 6.5 515 500

Example 26 Enzymatic Perhydrolysis of Triacetin Using a Combination ofLipase and Proteases at pH 4.0 and 6.5

Reaction mixtures were prepared containing 1 mg lipase (A “Amano” 12(Amano), Validase AN (Valley Research), or ICR-110 (BioCatalytics)), 20μL Alcalase® (Novozymes), and 20 μL Neutrase® (Novozymes) in 1.0 mL of50 mM buffer (either sodium acetate/acetic acid buffer at pH 4.0, orpotassium phosphate buffer at pH 6.5), additionally containing triacetin(250 mM) and hydrogen peroxide (500 mM). After stirring for 5 or 30minutes at 22° C., a 0.250-mL sample was filtered using a 30,000 NMWLfilter (Millipore UltraFree-MC) centrifuged for 2 minutes at 12,000 RPM.A portion of the filtered reaction samples was diluted 1:10 with ddwater and analyzed for hydrogen peroxide, and the remaining portion ofthe sample was directly analyzed for peracid using the HPLC assay method(Table 31). Reactions were also run with lipase alone or protease(s)alone for comparison of peracetic acid concentrations produced withoutthe combination of lipase and protease(s).

TABLE 31 Enzymatic Perhydrolysis of Triacetin (250 mM) using aCombination of Lipase and Proteases at pH 4.0 and 6.5. peraceticperacetic acid (ppm), acid (ppm), enzyme source pH 5 min 30 min noenzyme (control) 4.0 30 10 Alcalase ® and Neutrase ® 4.0 0 0 A“Amano”124.0 360 630 A“Amano”12/Alcalase ®/Neutrase ® 4.0 210 420 Validase AN 4.0310 540 Validase AN/Alcalase ®/Neutrase ® 4.0 160 380 ICR-110 4.0 300510 ICR110/Alcalase ®/Neutrase ® 4.0 270 470 no enzyme (control) 6.5 180310 Alcalase ® and Neutrase ® 6.5 240 480 A“Amano”12 6.5 490 800A“Amano”12/Alcalase ®/Neutrase ® 6.5 360 720 Validase AN 6.5 470 820Validase AN/Alcalase ®/Neutrase ® 6.5 460 780 ICR-110 6.5 340 540ICR110/Alcalase ®/Neutrase ® 6.5 490 560

Example 27 Enzymatic Perhydrolysis of a Mixture of Diacetin (250 mM),Triacetin (88 mM) and Monoacetin (192 mM) Using a Combination of Lipaseand Proteases at pH 4.0 and 6.5

Reaction mixtures were prepared containing 1 mg lipase (A “Amano” 12(Amano), Validase AN (Valley Research), or ICR-110 (BioCatalytics)), 20μL Alcalase® (Novozymes), and 20 μL Neutrase® (Novozymes) in 1.0 mL of50 mM buffer (either sodium acetate/acetic acid buffer at pH 4.0, orpotassium phosphate buffer at pH 6.5), additionally containing a mixtureof diacetin (250 mM), triacetin (88 mM) and monoacetin (192 mM) andhydrogen peroxide (500 mM). After stirring for 5 or 30 minutes at 22°C., a 0.250-mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) centrifuged for 2 minutes at 12,000 RPM. A portion of thefiltered reaction samples was diluted 1:10 with dd water and analyzedfor hydrogen peroxide, and the remaining portion of the sample wasdirectly analyzed for peracid using the HPLC assay method (Table 32).Reactions were also run with lipase alone or protease(s) alone forcomparison of peracetic acid concentrations produced without thecombination of lipase and protease(s).

TABLE 32 Enzymatic Perhydrolysis of a Mixture of Diacetin (250 mM),Triacetin (88 mM) and Monoacetin (192 mM) Using a Combination of Lipaseand Proteases at pH 4.0 and 6.5. peracetic peracetic acid (ppm), acid(ppm), enzyme source pH 5 min 30 min no enzyme (control) 4.0 20 10Alcalase ® and Neutrase ® 4.0 10 0 A“Amano”12 4.0 190 360A“Amano”12/Alcalase ®/Neutrase ® 4.0 120 260 Validase AN 4.0 150 270Validase AN/Alcalase ®/Neutrase ® 4.0 70 230 ICR-110 4.0 490 730ICR110/Alcalase ®/Neutrase ® 4.0 400 600 no enzyme (control) 6.5 90 170Alcalase ® and Neutrase ® 6.5 130 260 A“Amano”12 6.5 240 550A“Amano”12/Alcalase ®/Neutrase ® 6.5 250 560 Validase AN 6.5 280 440Validase AN/Alcalase ®/Neutrase ® 6.5 270 540 ICR-110 6.5 770 680ICR110/Alcalase ®/Neutrase ® 6.5 810 670

Example 28 Enzymatic Perhydrolysis of Triacetin Using a Protease at pH4.0 and 6.5

Reaction mixtures were prepared containing 5 mg protease (Savinase® 12T,Polarzyme® 12T or Everlase® 12T (Novozymes)) in 1.0 mL of 50 mM buffer(either sodium acetate/acetic acid buffer at pH 4.0, or potassiumphosphate buffer at pH 6.5), additionally containing triacetin (500 mM)and hydrogen peroxide (1000 mM). After stirring for 5 or 30 minutes at22° C., a 0.250-mL sample was filtered using a 30,000 NMWL filter(Millipore UltraFree-MC) centrifuged for 2 minutes at 12,000 RPM. Aportion of the filtered reaction samples was diluted 1:10 with dd waterand analyzed for hydrogen peroxide, and the remaining portion of thesample was directly analyzed for peracid using the HPLC assay method(Table 33).

TABLE 33 Enzymatic Perhydrolysis of Triacetin (500 mM) using a Proteaseat pH 4.0 and 6.5. peracetic acid peracetic acid enzyme source pH (ppm),5 min (ppm), 30 min no enzyme (control) 4.0 260 130 Savinase ® 12T 4.0220 170 Polarzyme ® 12T 4.0 420 40 Everlase ® 12T 4.0 80 380 no enzyme(control) 6.5 170 220 Savinase ® 12T 6.5 480 1240 Polarzyme ® 12T 6.5550 1940 Everlase ® 12T 6.5 710 1700

Example 29 Enzymatic Perhydrolysis of a Mixture of Diacetin (236 mM),Triacetin (83 mM) and Monoacetin (181 mM) Using a Protease at pH 4.0 and6.5

Reaction mixtures were prepared containing 5 mg protease (Savinase® 12T,Polarzyme® 12T or Everlase® 12T (Novozymes)) in 1.0 mL of 50 mM buffer(either sodium acetate/acetic acid buffer at pH 4.0, or potassiumphosphate buffer at pH 6.5), additionally containing a mixture ofdiacetin (236 mM), triacetin (83 mM) and monoacetin (181 mM) andhydrogen peroxide (500 mM). After stirring for 5 or 30 minutes at 22°C., a 0.250-mL sample was filtered using a 30,000 NMWL filter (MilliporeUltraFree-MC) centrifuged for 2 minutes at 12,000 RPM. A portion of thefiltered reaction samples was diluted 1:10 with dd water and analyzedfor hydrogen peroxide, and the remaining portion of the sample wasdirectly analyzed for peracid using the HPLC assay method (Table 34).

TABLE 34 Enzymatic Perhydrolysis of a Mixture of Diacetin (236 mM),Triacetin (83 mM) and Monoacetin (181 mM) Using a Protease at pH 4.0 and6.5. peracetic acid peracetic acid enzyme source pH (ppm), 5 min (ppm),30 min no enzyme (control) 4.0 10 130 Savinase ® 12T 4.0 40 100Polarzyme ® 12T 4.0 70 80 Everlase ® 12T 4.0 110 60 no enzyme (control)6.5 350 440 Savinase ® 12T 6.5 185 380 Polarzyme ® 12T 6.5 270 920Everlase ® 12T 6.5 290 760

1-82. (canceled)
 83. A biocidal composition comprising componentssuitable for producing peracid in situ, wherein said components compriseone or more prion-degrading enzymes, a suitable substrate, at least oneenzyme having perhydrolase activity, and a source of peroxygen.
 84. Thecomposition of claim 83, wherein said at least one enzyme havingperhydrolase activity is said one or more prion-degrading enzymes. 85.The composition of claim 84, wherein said one or more prion-degradingenzymes is at least one protease isolated from a host organism selectedfrom the group of Aspergillus saitoi, Rhizopus sp., Bacillus sp.,Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens,Bacillus lentus, Sus scrofa, Carica papaya, Ananas comosus, Streptomycesgriseus, and Tritirachium album.
 86. The composition of claim 85 whereinthe protease is selected from the group consisting of Pronase,Proteinase K, Alcalase and Neutrase.
 87. The composition of claim 86wherein the protease is selected from the group consisting of Alcalaseand Neutrase.
 88. The composition of claim 86 wherein said one or moreprion-degrading proteases is a mixture of at least two proteases. 89.The composition of claim 88 wherein said mixture of proteases comprisesPronase and Proteinase K.
 90. The composition of claim 89 wherein saidmixture of proteases comprises Alcalase and Neutrase.
 91. Thecomposition of claim 87, wherein the peracid produced in situ isperacetic acid.
 92. The composition of claim 91 wherein the suitablesubstrate used to produce peracetic acid in situ is selected from thegroup consisting of monoacetin, diacetin, triacetin, and mixturesthereof.
 93. The composition of claim 92 wherein the peracetic acidproduced in situ is at least 10 ppm.
 94. The composition of claim 88wherein the peracid produced in situ is peracetic acid.
 95. Thecomposition of claim 94 wherein the suitable substrate used to produceperacetic acid in situ is selected from the group consisting ofmonoacetin, diacetin, triacetin, and mixtures thereof.
 96. Thecomposition of claim 95 wherein the peracetic acid produced in situ isat least 10 ppm.
 97. The composition of claim 89 wherein the peracidproduced in situ is peracetic acid.
 98. The composition of claim 97wherein the suitable substrate used to produce peracetic acid in situ isselected from the group consisting of monoacetin, diacetin, triacetin,and mixtures thereof.
 99. The composition of claim 98 wherein theperacetic acid produced in situ is at least 10 ppm.
 100. The compositionof claim 90 wherein the peracid produced in situ is peracetic acid. 101.The composition of claim 100 wherein the suitable substrate used toproduce peracetic acid in situ is selected from the group consisting ofmonoacetin, diacetin, triacetin, and mixtures thereof.
 102. Thecomposition of claim 101 wherein the peracetic acid produced in situ isat least 10 ppm.